Ampk-related serine/threonine kinase, designated snark

ABSTRACT

The cloning and function of a new AMPK-related kinase, designated SNARK, are described. The kinase SNARK is involved in the stress response to glucose deprivation. Provided are the rodent and human genes encoding SNARK, and the SNARK protein and useful fragments, in isolated form. Also provided are SNARK expression systems and assays useful to identify SNARK substrates and SNARK activity modulators, and antibodies useful as SNARK modulators for instance in therapeutic applications to modulate the metabolism of glucose.

FIELD OF THE INVENTION

[0001] This invention is in the field of molecular biology and theemerging field of proteomics. It relates more particularly to certainnovel protein kinases, and their applications in drug discovery andmedical diagnostics.

BACKGROUND TO THE INVENTION

[0002] Protein kinase cascades are highly conserved between animals,fungi and plants. The Sucrose-Non Fermenting protein kinase (SNF1) fromSaccharomyces cerevisiae and its mammalian counterpart, AMP-activatedprotein kinase (AMPK), form a family of serine/threonine kinases andrepresent key components in yeast and mammalian stress response systems[1]. This family of kinases is commonly activated in response tocellular and environment stresses, including nutrient deprivation. SNF1responds to glucose deprivation by derepressing genes implicated incarbon source utilization and by modulating transcription ofglucose-regulated genes involved in gluconeogenesis, respiration,sporulation, thermotolerance, peroxisome biogenesis, and cell cycleregulation [2]. AMPK is similarly activated by environmental stressesthat result in increases in the cellular ATP:AMP ratio. Activated AMPKswitches off anabolic pathways (e.g. fatty acid and cholesterolsynthesis) and induces ATP-generating catabolic pathways (i.e. fattyacid oxidation) [1].

[0003] The SNF1/AMPK family of serine/threonine kinases has expandedrapidly following the cloning of several SNF1/AMPK-related proteinkinases in plants [3-11], Plasmodium falciparum[12], Chlamydomonas [13]and mammals [14-19]. These protein kinases have been assigned to theSNF1/AMPK family primarily on the basis of their structural similaritywith the catalytic domains of SNF1 and AMPK. The available structuraland functional data is consistent with the notion that SNF1, AMPK andrelated kinases represent components of signaling cascades that controlmetabolism, gene expression and perhaps cell proliferation in responseto cellular, metabolic and environmental stress [20].

[0004] In general, it is known that the chemical AICAR(5-aminoimidizole-4-caroximide riboside) activates AMPK, and adownstream set of metabolic responses that may be insulin-like incertain tissues such as muscle and liver. The AMPK system seems tofunction as a cellular fuel gauge, monitoring the energy state of thecell and protecting the cell from energy depletion. It is not clear inthe literature that AMPK is responsible for all of the downstream eventsdetected following AICAR treatment of cells. The system is often studiedby treating cells with AICAR, which mimics the effects of AMP on theAMPK system. AICAR was previously presumed to be relatively specific foractivation of AMPK. Given the central importance of the AMPK cascade infuel metabolism and energy balance, and given the numerous medicalconditions and disorders that manifest from an imbalance in thiscascade, it would be desirable to expand the understanding of thosecomponents that are critical for its proper functioning.

SUMMARY OF THE INVENTION

[0005] There has now been identified a novel mammalian member of thekinase family, designated SNARK, having significant homology to thecatalytic domain of the SNF1/AMPK family of serine threonine proteinkinases. Results presented herein identify SNARK as a novel glucose- andAICAR-regulated mammalian member of the AMPK-related kinase gene family,and confirm that SNARK represents a new candidate mediator of thecellular responses to metabolic stress.

[0006] In one of its aspects, the present invention provides an isolatedpolynucleotide, in the form of RNA or DNA, including cDNA and genomicDNA and synthetic equivalents thereof, that encodes a SNARK proteinwhich is the SNARK protein of SEQ ID NO. 1, or a mammalian homologthereof including the human SNARK protein encoded within humanchromosome 1q32, or a variant or chimeric SNARK protein that retainsSNARK activity and shares at least 70%, e.g. 80% and more preferably atleast 90% e.g. 95-99%, sequence identity with the SNARK protein of SEQID NO. 1.

[0007] Also provided by the present invention are polynucleotides andoligonucleotides that hybridize with the SNARK-encoding polynucleotides.Such hybridizing poly- and oligonucleotides are optionally detectablylabeled, and have a sequence complementary to, or an anti-sense versionof, a characterizing region of the SNARK-encoding polynucleotides. Suchhybridizing poly- and oligonucleotides are useful to detectSNARK-encoding polynucleotides in a given sample, for instance to probefor or amplify SNARK-encoding mRNA or DNA in a library, or to identifySNARK-encoding polynucleotide in a given tissue sample by in situlocalization. Such hybridizing poly- and oligonucleotides are usefulalso to arrest endogenous expression of the SNARK-encodingpolynucleotide, for instance to modulate SNARK production fortherapeutic intervention. In embodiments of the invention, theoligonucleotides are designed to bind to the polynucleotide regionencoding the C-terminal region of the SNARK protein, which among othermembers of the SNF1/AMPK family, comprises unique protein sequencedistinctive of SNARK. Thus, in embodiments of the invention, there areprovided oligonucleotides that hybridize under stringent conditions withthat region of SNARK-encoding DNA that codes for a unique C-terminalregion thereof, such as the region constituted by amino acids 310through 630 of SEQ ID NO. 1 or the comparable region of a homologthereof, or a sub-region comprising at least about 20 nucleotides, e.g.,desirably about 40 nucleotides, thereof.

[0008] The polynucleotides of the present invention are useful, inanother aspect, for expression to produce SNARK protein in isolatedform, or as a protein conjugate. Accordingly, there are provided vectorsthat incorporate the SNARK-encoding polynucleotides in operablecombination with expression controlling elements for driving theexpression thereof in a suitable host. In related aspects of theinvention, there are provided cellular hosts incorporating theexpressible, SNARK-encoding polynucleotides. Also provided are methodsfor SNARK production, which comprises the step of culturing SNARKproduction hosts under conditions adapted for producing SNARK. In afurther related aspect, the SNARK production hosts are useful to screenfor modulators of SNARK activity, thereby to identify agents useful tomodulate SNARK activity either in vitro, or in vivo for therapeuticpurposes.

[0009] Included among the oligonucleotides useful as probes to identifySNARK homologs are the human EST sequences reported in the BLASTdatabase as having homology with a region of SEQ ID NO. 2 that is atleast about 85%. Such oligonucleotides include those referenced asgb/AI469033.1/AI469033 (ti70a02.x1) reported as NCI_CGAP_Kid11 (whichscores 525 bits at an E value of e-146); and as gb/AA995360.1/AA995360(or74b03.s1) reported as NCI_CGAP_Lu5 (which scores 426 bits at an Evalue of e-117). The present invention thus embraces the human homologof SNARK, which human homolog incorporates amino acid sequence that isencoded by such human EST sequences. The present invention furtherembraces polynucleotides that encode the human homolog of SNARK, andincorporates amino acid sequence encoded by such ESTs or sequence havingat least about 95% identity therewith as exemplified further herein. Ina related aspect, the present invention further provides a method fordetecting SNARK-encoding DNA polynucleotide in a sample, in which suchhuman ESTs, and extended or fragmented forms thereof are used optionallyin labeled forms as probes.

[0010] In another of its aspects, the present invention provides SNARKprotein, in isolated form, and optionally incorporating a detectablelabel. Such SNARK protein may be in the form of the rat SNARK protein ofSEQ ID NO. 1, a mammalian homolog thereof including human SNARK andmouse SNARK, variants of such mammalian forms of SNARK, and chimericforms thereof in which regions or domains thereof, such as the catalyticdomain, have been exchanged. The SNARK protein may further comprise acarrier useful, for instance, to raise antibodies thereto.Alternatively, the SNARK protein may be combined with a pharmaceuticallyacceptable carrier, for use as a therapeutic.

[0011] Also provided by the present invention are SNARK fragments, e.g.,having N- and/or C-terminal truncations, including for instance animmunogenic fragment against which antibodies can be raised, orcomprising a region capable of inhibiting the binding of SNARK with itsbinding partners that participate in the signaling cascade in whichSNARK is involved endogenously, thereby to downregulate SNARK activity.Such immunogenic fragments comprise at least about 20 amino acids, andpreferably incorporate a contiguous portion of the C-terminal SNARKregion spanning residues 310 through 630 of SEQ ID NO. 1 or acorresponding region of a mammalian homolog of SEQ ID NO. 1., or avariant of such region having one or more, e.g. up to about 10,conservative amino acid substitutions.

[0012] Also provided, in another aspect of the present invention, areantibodies that bind selectively to SNARK,e.g., with preference relativeto other AMP kinases. In embodiments, these SNARK-binding antibodies arein detectably labeled form, to allow for the detection of SNARK in agiven sample. Alternatively, the SNARK antibodies are used to modulateSNARK activity either in vivo, for therapeutic purposes, or in vitro,for drug screening and related investigational purposes.

[0013] In another of its aspects, the present invention provides amethod for assaying SNARK activity, the method comprising the step ofobtaining a candidate SNARK protein, incubating the SNARK protein with aSNARK substrate under phosphorylating conditions, and then determiningwhether phosphorylation has occurred, wherein phosphorylation revealsthe SNARK candidate protein has SNARK activity. In a related aspect ofthe invention, the assay is modified to identify SNARK activitymodulators, in which SNARK protein, a selected SNARK substrate and acandidate modulator of SNARK activity are incubated underphosphorylating conditions, and determining the extent ofphosphorylation in the presence of the candidate modulator relative tothe extent of phosphorylation in the absence of the candidate modulator,wherein modulating activity is revealed by a difference inphosphorylation in the presence of the modulator relative to the absenceof the modulator.

[0014] These and other aspects of the invention are described in greaterdetail with reference to the accompanying drawings, in which:

REFERENCE TO THE DRAWINGS

[0015]FIG. 1(A). Southern blot analysis of genomic DNA isolated from ratliver and human lymphocytes. Genomic DNAs (15 μg) were digested with oneof the restriction endonucleases BamHI, EcoRI or HindIII. The blot washybridized with a 1.5 kb fragment (≈nt 1400-2929) corresponding largelyto the 3′-end of the SNARK protein. The approximate positions of the DNAsize markers are indicated at the right. (B). Chromosomal localizationof human SNARK. A 450 bp fragment of the rat UV126 cDNA [20] was used asa probe to screen a P1-derived artificial chromosome (PAC) library. Theprobe identified one genomic PAC as positive and this PAC was mapped tohuman chromosome 1q32. Positive hybridization signals at 1q32 (seen asbright spots on the chromosome) were noted on both homologues in >90% ofthe cells.

[0016]FIG. 2. Nucleotide and deduced amino acid sequences of rat SNARK.The deduced amino acid sequence of SNARK (SEQ ID NO. 1) is shown insingle-letter code above the respective coding nucleotide sequence (SEQID NO. 2). Nucleotide number assignment is listed on the right and aminoacid number assignment is shown as underlined numbers on the right. Theprotein serine/threonine kinase catalytic domains are boxed. The proteinkinase ATP-binding region signature is underscored with a dotted line.The serine/threonine kinase active-site signature is underlined with adashed line.

[0017]FIG. 3. Alignment of the deduced amino acid sequence of rat SNARKand other members of the SNF1/AMPK family using the CLUSTAL W algorithm.The protein kinase catalytic domains are boxed. Identical residues areindicated by asterisks and conservative substitutions are indicated bydots under the sequences (‘:’ indicates substitution with a stronggroup, score >0.5, and ‘.’ indicates a substitution with a weak group,score ≦0.5). The amino acid residues are numbered on the right.

[0018]FIG. 4(A). Northern blot analysis of rat tissues showing tissuedistribution of SNARK mRNA. Ten μg of total RNA isolated from each rattissue (indicated above the appropriate lane) was electrophoresed,transferred to a nylon membrane and probed with a fragment of theSNARK/pcDNA3.1 corresponding to the protein coding region (nt 0-1975)and exposed to film. Positions of the 28S and 18S ribosomal RNA areindicated at the right of the autoradiograph. (B). Reverse transcriptasePCR analysis of SNARK in various rat tissues. The upper panel shows anethidium bromide stained gel of RT-PCR products resulting fromfirst-strand cDNAs prepared from rat heart (lane 1), skin (lane 2),spleen (lane 3), kidney (lane 4), lung (lane 5), liver (lane 6), uterus(lane 7), testis (lane 8) and NRKC cells (lane 9). A negative controlreaction containing no first-strand DNA was included to verifyspecificity of primer products (lane 10). The lower panel shows aSouthern analysis of the RT-PCR products resulting from each tissuetype. The Southern blot was hybridized with a fragment of SNARKcorresponding to its protein-coding region (nt 0-1975). Positions ofmigration of the DNA size markers are shown on the right of the figure.

[0019]FIG. 5(A) Analysis of SNARK protein transcribed and translated inrabbit reticulocyte lysate. In lanes 1 and 2, one-tenth of the TNTreaction was loaded directly onto a 8% polyacrylamide-SDS gel. Lanes 3-5are size-fractionated immunoprecipitation (IP) reactions. Lanes 3 and 4are control IP reactions where no TNT products or T7-luciferase TNTproducts were incubated with SNARK antiserum #14, respectively. Lane 5shows SNARK TNT immunoprecipitated with SNARK antiserum. Following gelelectrophoresis, the SDS-polyacrylamide gel was fixed, dried and exposedto BioMax MS film with intensifying screen for 4 hours. (B) Westernanalysis of SNARK protein in stably transformed BHK cells. NRKC cellextract (750 μg; lane 1) or 500 μg of BHK+1 (lanes 2 and 3) and BHK+11(lane 4) were immunoprecipitated with SNARK antiserum #14 (lanes 1, 2and 4) or with nonimmune serum (lane 3), electrophoresed on a 8%polyacrylamide-SDS and transferred onto a PVDF membrane. Westernanalysis was performed using the SNARK antiserum #16. The positions ofthe protein standards are shown at the right in kDa. The position of theSNARK protein is listed at the left of the autoradiograph.

[0020]FIG. 6. Autophosphorylation of SNARK. SNARK was immunoprecipitatedfrom 500 μg of wildtype BHK, BHK+1 and BHK+11 cell extract with eitherSNARK antiserum #14 (lanes 1, 2, 4 and 6) or nonimmune serum (lanes 3and 5). Lane 6 is a negative immunoprecipitation control containing 500μg of BHK+1 cell extract but no antiserum. Immunoprecipitates wereincubated with [γ]-ATP³² at 30° C. and the reactions were stopped after30 minutes by the addition of 2X SDS loading buffer and boiling for 5minutes. Samples were electrophoresed on a 8% polyacrylamide-SDS gel andthe gel was dried and exposed to BioMax MS film.

[0021]FIG. 7. The SNARK protein possesses AMPK-like phosphotransferaseactivity. SNARK protein was immunoprecipitated from 500 μg of celllysate from wildtype BHK, BHK−1, BHK+1 and BHK+11 cells. Kinase assayswere performed using the 200 μM SAMS peptide as substrate in kinasereaction cocktail. NRKC cells (stippled box); wildtype BHK cells (solidbox); BHK+1 and +11 cells (hatched boxes). Results are the means±S.E.M.for 2 experiments (n=5 each time). For comparative purposes, AMPKα2activity was assayed in these cell lines and found to be equivalent to,4-fold lower and 1.5-fold lower than the SNARK activity levels measuredunder basal growth conditions in wildtype BHK, SNARK-transfected BHK andNRKC cell lines, respectively (data not shown).

[0022]FIG. 8.(A) Activation of SNARK in NRKC cells using AICAR. NRKCcells were treated with 0, 0.5 mM, 1 mM or 2 mM AICAR for 1 hour. SNARKprotein was immunoprecipitated from 500 μg of cell lysates with SNARKantiserum and kinase assays were performed in the presence of 200 μM AMPusing the SAMS peptide. Solid box represents control (0 mM AICAR)samples and hatched boxes represent AICAR-treated samples. Data isexpressed as phosphotransferase activity relative to control values(control=1) and represents the means±S.E.M. for 2 individual experimentswith at least 10 samples per group per experiment. *p<0.06, relative tocontrol values. Using the same assay conditions, basal AMPK-α2 activitywas found to be 1.5-fold lower than SNARK activity and increased atleast 2-fold upon treatment with ImM AICAR in NRKC cells (data notshown). Basal AMPKα1 activity was found to be 88 times higher than basalSNARK activity, but was not stimulated by treatment with AICAR (data notshown). (B) Activation of SNARK in wildtype BHK cells resulting fromglucose deprivation. Wildtype BHK cells were exposed to glucose-freemedium for 0 or 90 minutes. SNARK was immunoprecipitated and kinaseassays were performed using the SAMS peptide as substrate in thepresence of AMP Solid box represents control (25 mM glucose) samples andhatched boxes represent glucose-deprived samples. Results are expressedas phosphotransferase activity relative to control values (control=1)and represents the mean±S.E.M. from 2 individual experiments with atleast 10 samples per group per experiment. *p<0.03, relative to controlvalues. Basal AMPKα2 activity levels measured in wildtype BHK cells werecomparable with basal SNARK activity detected in these cells (data notshown).

[0023]FIG. 9 compares, at the amino acid level, the 1-251 region of ratSNARK with (A) the amino acid sequence encoded by a human polynucleotideand (B) the amino acid sequence of a mouse polynucleotide, both of whichwere identified by in silico screening. Provided at the top of eachFigure are the accession numbers for the identified homologs, andcorresponding literature citations where available.

[0024]FIG. 10 provides a comparison of rat SNARK-encoding DNA withpolynucleotides of human genomic, RNA or cDNA origin identified insilico by searching (1) with the complete sequence of rat SNARK-encodingcDNA, including (A) correlations with the human genome database, withsequence gaps determined by sequencing of SNARK-encoding DNA isolatedfrom the human cell line HaCaT, (B) correlations with the human mRNAdatabase; and (2) with the amino acid sequence of rat SNARK proteinincluding (C) correlations with the human EST database. FIG. 10 (D)provides a comparison of rat SNARK encoding DNA with the sequenceencoding human SNARK cloned from the HACAT cell line. Provided at thetop of each Figure are the accession numbers for the identifiedhomologs, and corresponding literature citations where available.

[0025]FIG. 11 provides a comparison of rat SNARK-encoding DNA withpolynucleotides of mouse genomic, RNA or cDNA origin identified insilico by searching (1) with the complete sequence of rat SNARK-encodingcDNA, including (A) correlations with the mouse high throughput genomedatabase; (B) correlations with the mouse nr database; and (2) with theamino acid sequence of rat SNARK protein including (C) and (D)correlations with the mouse EST database. FIG. 11(E) provides acomparison of rat SNARK encoding DNA with the sequence encoding mouseSNARK cloned from hairless mice. Provided at the top of each Figure arethe accession numbers for the identified homologs, and correspondingliterature citations where available.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0026] The present invention relates to a novel mammalian form of akinase related to the family of SNF1/AMPK serine/threonine proteinkinases, which has been designated SNARK. In embodiments, the SNARKprotein is provided in “isolated” form, i.e., in a form essentially freefrom proteins with which that form of SNARK is normally associated.

[0027] As illustrated in FIG. 2, the rat form of the SNARK proteincomprises 630 amino acids [SEQ ID NO. 1]. Identified on FIG. 2 areconsensus regions indicating the protein serine/threonine kinasecatalytic domains (boxed), the protein kinase ATP-binding regionsignature (underscored with a dotted line), and the serine/threoninekinase active-site (underlined with a dashed line).

[0028] The alignment of the deduced amino acid sequence of rat SNARK andother members of the SNF1/AMPK family using the CLUSTAL W algorithm isshown in FIG. 3. The protein kinase catalytic domains are boxed.Asterisks indicate identical residues and conservative substitutions areindicated by dots under the sequences. The amino acid residues arenumbered on the right. Accession numbers of the respective comparisonsequences are: p78 (PIR, s27966), emk (PIR, s31333), SIK (gb, AB020480),SNF1 (PIR, a26030) and AMPK (gb, z29486).

[0029] Thus, in one embodiment, the invention encompasses the SNARKprotein identified by SEQ ID NO. 1. The invention also encompasses SNARKhomologs, including the human homolog and the mouse homolog, andvariants and chimeric forms of SNARK, which retain functional activityof SNARK. Variants of SNARK include SNARK proteins that differ relativeto a SNARK homolog by incorporating amino acid substitutions,insertions, or deletions that do not disrupt SNARK function, such asSNARK phosphorylating activity. Generally, such alterations will notaffect more than about 10% of the primary structure of SNARK. Forinstance, amino acid substitutions, deletions or insertions will notgenerally involve more than about 20 amino acids, e.g., more than about10 amino acids. In embodiments of the invention, the SNARK homolog orvariant shares at least 70%, e.g., at least 80% identity to the SNARKhaving SEQ ID NO. 1. The C-terminal region of SNARK is a particularlyunique region of the protein, having almost no homology in its 310-630region with the similarly positioned regions of other members of theSNF1/AMPK family, as shown in FIG. 3. Accordingly, in a preferredembodiment, variants of the SNARK protein share at least 90% identity,and more preferably at least 95%, e.g., 98%-99% identity, with this 310-630 region of SNARK. In this context, a preferred SNARK variant is onehaving the noted identity with the C-terminal SNARK region, and anoverall identity of at least 80%, and more preferably 90%, amino acidsequence identity to the SNARK of SEQ ID NO. 1. A most preferred SNARKvariant is one having at least 95% amino acid sequence identity thereto.It is recognized that mammalian SNARK proteins may exhibit a greaterdegree of amino acid variation in the C-terminal region of the SNARKprotein, compared to the more conserved functional N-terminal kinasedomains.

[0030] Proteins that exhibit “SNARK activity” are defined as thoseproteins that (1) are recognized by antisera against native rodent orhuman SNARK, (2) exhibit phosphorylating activity against a synthetic ornatural substrate, such as SAMS, that is also recognized by nativerodent or human SNARK, and (3) exhibit the following rank order ofsubstrate selectivity: SAMS>MBP>B-casein>whole histonefraction>protamine sulfate. Such proteins, that exhibit SNARK activitycan be further characterized structurally as exhibiting at least 75%amino acid identity, and more desirably at least 90% identity within theconserved functional kinase domains of rat SNARK as outlined in FIG. 2and 3.

[0031] In embodiments of the present invention, the SNARK protein is ahuman homolog of SEQ ID NO. 1. In embodiments, the human homologincorporates amino acid sequence encoded by highly homologous(Value>200) EST and genomic clones identifiable in the public BLAST orsimilar database upon searching against the rat SNARK DNA of SEQ ID NO.2. Examples of such human EST's are those reported in GenBank asaccession numbers AI469033.1 and AA995360.1, and available as IMAGEclones 2137322 and 1601549, respectively. The sequences of such clonesare mapped onto SNARK-encoding DNA, in FIG. 10. In a particularembodiment of the invention, the SNARK protein is a human homolog thatincorporates, within its overall sequence, the 251 amino acid sequencedepicted in FIG. 9A, or a variant thereof incorporating amino acidalteration(s) that does not disrupt SNARK activity. In other specificembodiments, the human SNARK protein incorporates amino acid sequencesthat are encoded by one or more of the coding regions of thepolynucleotides shown in FIGS. 10A, 10B, 10C and 10D.

[0032] The human SNARK protein is characterized by encoded genesequences that are identified in public domain or proprietary databases,and is recognized by antisera directed at conserved domains withinrodent and human SNARK. The human SNARK protein is expected to exhibit75%, and suitably, 90% amino acid identity with rodent SNARK at keyfunctional kinase domains, as exemplified by sequence alignment shown inFIG. 9. A human SNARK protein also exhibits the same degree of substratespecificity exhibited by rat SNARK, as shown in Table 1, infra.

[0033] Obtaining of the nucleotide sequence of an intact and full lengthcDNA encoding such a human homolog is alternatively achieved byscreening a suitable cDNA library, such as a cDNA library generated fromkeratinocytes obtained for instance from skin or muscle tissue such asheart, or from the HaCaT cell line in the manner exemplified herein. Thetissue desirably is first irradiated in the manner reported by Rosen etal [20], to induce human SNARK expression. Screening of the library isachieved either by labeled probing with the SNARK SEQ ID NO. 2, or witha sequence corresponding to the ESTs and genomic clones just described,or with any major hybridizing fragment thereof. Alternatively, the humanSNARK homologue may be obtained by using the rat SNARK sequence providedherein to screen human DNA databases to identify previously unknownnucleotide sequences that can be identified as human SNARK homologues.As one example of this possibility, we identify human EST's reported inGenBank as accession numbers AI469033.1 and AA995360.1, that correspondto cDNAs encoding partial human SNARK coding sequences. The remainder ofthe human SNARK sequences can be obtained by cDNA cloning, RT-PCR, orfurther additional database searches, using the information providedherein. More particularly, the sequences encoding regions of human SNARKare provided in FIG. 10.

[0034] Desirably, the SNARK variants retain the property ofautophosphorylation possessed by SNARK. Such SNARK activity can beassessed using the autophosphorylation assay herein described.

[0035] As noted, the invention further embraces homologs of the SNARKprotein identified in SEQ ID NO. 1 including the human homolog encodedon chromosome site 1q32, and other mammalian homologs encoded bypolynucleotides that hybridize under stringent conditions with the SNARKof SEQ ID NO. 2.

[0036] In another embodiment, the invention provides the mouse homologof the rat SNARK of SEQ ID NO. 1. that incorporates, within its overallsequence, the 251 amino acid sequence depicted in FIG. 9B, or a variantthereof incorporating amino acid alterations that do not disrupt SNARKactivity. In more specific embodiments, the mouse SNARK proteinincorporates amino acid sequences that are encoded by one or more of thenon-rat coding regions of the polynucleotides shown in FIGS. 11A through11E. The mouse SNARK protein also exhibits 75%, and suitably 90% aminoacid identity with rat SNARK at key functional kinase domains, asexemplified by sequence alignment shown in FIG. 9. A mouse SNARK proteinalso exhibits the same degree and rank order of substrate specificity ascharacterized for rat SNARK, as shown in Table 1, infra.

[0037] The invention also relates to chimeric versions of the SNARKprotein. Such chimeric SNARK proteins are hybrid SNARK proteins in whicha selected region of a given SNARK protein has been replaced, orexchanged, by a corresponding region from a SNARK homolog. Such regionssuitable for exchange include, for instance, the kinase catalyticdomain, or the ATP-binding region or the serine/threonine kinase activesite, all of which are shown in FIG. 2 for rat SNARK. Such chimericSNARK proteins expectedly retain SNARK activity, yet allow such functionto be assessed in different SNARK backgrounds, if desired. Accordingly,in embodiments of the invention, there are provided chimeric SNARKproteins in which a functional domain or region of one form of SNARK isreplaced by a corresponding region from a SNARK homolog.

[0038] The invention also embraces fragments of SNARK, includingfragments of the SNARK of SEQ ID NO. 1 ,and homologous counterpartfragments of human and mouse SNARK including those shown in FIGS. 9A and9B respectively, that are useful for various purposes. In oneembodiment, the invention includes immunogenic fragments, thatincorporate at least about 5, e.g., at least about 20 contiguous aminoacids, and suitably up to about 200 amino acids or more corresponding toSNARK epitopes, including for instance regions of such length within theC-terminal region thereof, spanning for instance amino acids 310-630 ofrat SNARK and corresponding regions of homologs such as human and mouseSNARK. Alternatively, such fragments may have an amino acid sequencethat is encoded by the ESTs just described, or particularly by the humanand mouse polynucleotides shown in FIGS. 10 and 11.

[0039] The invention also encompasses polynucleotides which encode SNARKor which encode SNARK variants or chimeras. Accordingly, any nucleicacid sequence which encodes the amino acid sequence of SNARK, andvariants and chimerics thereof, can be used to produce recombinantmolecules which express SNARK proteins. In a particular embodiment, theinvention encompasses a polynucleotide consisting of a nucleic acidsequence illustrated as the SNARK-encoding region in FIG. 2, anddesignated herein as SEQ ID NO. 2 (i.e., nucleotides 83-1975).

[0040] Also embraced by the present invention are polynucleotides thatencode human SNARK. Such polynucleotides include those partial sequencesshown in FIG. 10 as having homology with the coding region of rat SNARK.

[0041] The present invention also includes polynucleotides that encodemouse SNARK. Such polynucleotides include those partial sequences shownin FIG. 11 as having homology with the coding region of rat SNARK.

[0042] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding SNARK, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring SNARK.

[0043] Although nucleotide sequences which encode SNARK and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring SNARK under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding SNARK or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding SNARK and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0044] The invention also encompasses production of DNA sequences, orfragments thereof, which encode SNARK and its fragments, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding SNARK or any fragment thereof.

[0045] Also encompassed by the invention are polynucleotide sequences,and especially full length SNARK-encoding sequences, that are capable ofhybridizing to (1) the SNARK of SEQ ID NO. 2, or to the polynucleotidesshown in FIGS. 10 and 11 as representing homologous regions within thehuman and mouse genes encoding SNARK, respectively, or to thecomplements thereof, under various conditions of stringency as taught inWahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511). “Stringentconditions” or “stringency” refers to conditions that allow for thehybridization of substantially related nucleic acid sequences. Forinstance, such conditions will generally allow hybridization of sequencewith at least about 85% sequence identity, preferably with at leastabout 90% sequence identity, more preferably with at least about 95%sequence identity. Polynucleotides that encode full lengthSNARK-encoding sequences are those which, upon expression, yield aprotein having one or more SNARK activities, includingautophosphorylation and response to AICAR.

[0046] The polynucleotides encoding SNARK may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA, such as the 1q32 regionto which human SNARK has been mapped, is first amplified in the presenceof primer to a linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0047] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68.degree.-72.degree. C. (actually between 53C(mouse)-72 C(extension) for SNARK). The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0048] Another method which may be used to locate SNARK homologs is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. Alternatively,and as noted above, commerciallyavailable and now routine software and search engines can be used tosearch public databases of nucleic acid and polypeptide databases toidentify homologous sequences that are likely, by closely matchedsequence identities, e.g., to have SNARK activity.

[0049] When screening for full-length cDNAs for instance to find fulllength SNARK homologs, it is preferable to use libraries that have beensize-selected to include larger cDNAs. Also, random-primed libraries arepreferable, in that they will contain more sequences that contain the 5′regions of genes. Use of a randomly primed library may be especiallypreferable for situations in which an oligo d(T) library does not yielda full-length cDNA. Genomic libraries may be useful for extension ofsequence into 5′ non-transcribed regulatory regions.

[0050] The screening for further SNARK homologs can be achieved byapplying standard hybridization or amplification techniques to atissue-derived polynucleotide library. A wide variety of such librariesare commercially available. Where construction of a cDNA library isnecessary, established techniques are applied. For example, isolation ofa SNARK homolog typically will entail extraction of total messenger RNAfrom a fresh source of tissue. In this respect, it is noted that asingle copy of the SNARK gene appears to be expressed in all tissues,although the testes and certain other tissues carry internally deletedforms thereof. Following conversion of message to cDNA, the library canbe formed in for example a bacterial plasmid, more typically abacteriophage. Such bacteriophage harboring fragments of the DNA aretypically grown by plating on a lawn of susceptible E. coli bacteria,such that individual phage plaques or colonies can be isolated. The DNAcarried by the phage colony is then typically immobilized on anitrocellulose or nylon-based hybridization membrane, and thenhybridized, under carefully controlled conditions, to a radioactively(or otherwise) labelled probe sequence to identify the particular phagecolony carrying the DNA insert of particular interest, in this case ahomolog of rat SNARK. The phage carrying the particular gene of interestis then purified away from all other phages from the library, in orderthat the foreign gene may be more easily characterized. Typically, thegene or a portion thereof is then isolated by subcloning into aplasmidic vector for convenience, especially with respect to the fulldetermination of its DNA sequence.

[0051] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterSNARK encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0052] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding SNARK may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for modulators, i.e., inhibitors or activators ofSNARK activity, it may be useful to encode a chimeric SNARK protein thatcan be recognized by a commercially available antibody. A fusion proteinmay also be engineered to contain a cleavage site located between theSNARK encoding sequence and the heterologous protein sequence, so thatSNARK may be cleaved and purified away from the heterologous moiety.

[0053] In another embodiment, sequences encoding SNARK and variant andchimeric forms thereof may be synthesized, in whole or in part, usingchemical methods well known in the art (see Caruthers, M. H. et al.(1980) Nucl. Acids Res. Symp. Ser. 7:215-223; Horn, T. et al. (1980)Nucl. Acids Res. Symp. Ser. 7:225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of SNARK, or a fragment thereof. For example, peptidesynthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A peptidesynthesizer (Perkin Elmer).

[0054] In another aspect of the invention, polynucleotide sequences orfragments thereof which encode SNARK, its variants, chimerics andfragments of these, may be used in recombinant DNA molecules to directtheir expression in appropriate host cells. In order to express abiologically active SNARK, the nucleotide sequences encoding SNARK or avariant or chimeric thereof, may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

[0055] Methods that are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding SNARKand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0056] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding SNARK. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0057] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding SNARK,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0058] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for SNARK. For example, whenlarge quantities of SNARK are needed for the induction of antibodies,vectors that direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), in which the sequence encodingSNARK may be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of .beta.-galactosidaseso that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. PGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0059] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0060] An insect system may also be used to express SNARK. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding SNARKmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of SNARK will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which SNARK may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0061] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding SNARK may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable virusthat is capable of expressing SNARK in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0062] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10 M are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

[0063] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding SNARK. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding SNARK, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0064] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. Differenthost cells which have specific cellular machinery and characteristicmechanisms for post-translational activities (e.g., BHK, CHO, HeLa,MDCK, HEK293, and W138), are available from the American Type CultureCollection (ATCC; Bethesda, Md.) and may be chosen to ensure the correctmodification and processing of the foreign protein.

[0065] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress SNARK may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0066] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk.sup.- oraprt.sup.-cells, respectively. Also, antimetabolite, antibiotic orherbicide resistance can be used as the basis for selection; forexample, dhfr which confers resistance to methotrexate (Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confersresistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin,F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Murry, supra). Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, .beta. glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121-131).

[0067] Alternatively, host cells that contain the nucleic acid sequenceencoding SNARK and express SNARK may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein. The presence of polynucleotide sequencesencoding SNARK can be detected by DNA-DNA or DNA-RNA hybridization oramplification using probes or fragments or fragments of polynucleotidesencoding SNARK. Nucleic acid amplification based assays involve the useof oligonucleotides or oligomers based on the sequences encoding SNARKto detect transformants containing DNA or RNA encoding SNARK.

[0068] A variety of protocols for detecting and measuring the expressionof SNARK, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson SNARK is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0069] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding SNARKinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding SNARK, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0070] Host cells transformed with nucleotide sequences encoding SNARKmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode SNARK may be designed to contain signal sequences which directsecretion of SNARK through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding SNARK tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and SNARK may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingSNARK and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying SNARK from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0071] In addition to recombinant production, fragments of SNARK may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of SNARK may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0072] Animal model systems which elucidate the physiological andbehavioral roles of the SNARK are produced by creating transgenicanimals in which the activity of SNARK is either increased or decreased,or the amino acid sequence of the expressed SNARK is altered, by avariety of techniques. Examples of these techniques include, but are notlimited to: 1) Insertion of normal or mutant versions of DNA encodingSNARK, by microinjection, electroporation, retroviral transfection orother means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal or 2)Homologous recombination of mutant or normal, human or animal versionsof these genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of the SNARK sequences.The technique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and so is useful forproducing an animal that cannot express native SNARK but does express,for example, an inserted mutant SNARK, which has replaced the nativeSNARK in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome,but does not remove them, and so is useful for producing an animal thatexpresses endogenous and exogenous SNARK, to elicit its over-expression.

[0073] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium such as M2 medium. DNA or cDNAencoding SNARK is cesium chloride purified from a vector by methods wellknown in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the transgene.

[0074] In another of its aspects, the present invention providesantibodies that bind to SNARK. Antibodies to SNARK may be generatedusing methods that are well known in the art. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments, and fragments produced by a Fab expressionlibrary. Neutralizing antibodies, (i.e., those which inhibit dimerformation) are especially preferred for therapeutic use.

[0075] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith SNARK or with a SNARK variant or chimeric, or any fragment oroligopeptide thereof which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

[0076] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to SNARK have an amino acid sequenceconsisting of at least five amino acids and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Preferred antibodies are those raised against amino acid sequences ofthe SNARK protein that are unique and which do not exhibit 100% identitywith the amino acid sequences of other proteins, as determined bycomputer-based searching of biological databases, for instance. Shortstretches of SNARK amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule. Examples of useful SNARK fragments includecontiguous regions of at least 5, more desirably at least 10 amino acidsand especially from 100 or about 200 amino acids within the C-terminalregion of rat SNARK from residue 310 to residue 630 , or correspondingregions within SNARK homologs including human and mouse SNARK.Particularly useful fragments are those that correspond to the activesite of the kinase catalytic domains I-XI, outlined in FIG. 3, as wellas the ATP binding domain (amino acids 63-89) and the active sitesignature motif, (aa 175-187). (FIG. 2) and variants that share at leastabout 95% identity therewith.

[0077] Monoclonal antibodies to SNARK may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0078] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceSNARK-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0079] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299). Antibodyfragments that contain specific binding sites for SNARK may also begenerated. For example, such fragments include, but are not limited to,the F(ab′)2 fragments which can be produced by pepsin digestion of theantibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-1281).

[0080] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificity are well known inthe art. Such immunoassays typically involve the measurement of complexformation between SNARK and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering SNARK epitopes is preferred, but a competitivebinding assay may also be employed.

[0081] Chemical and structural homology exists among the protein kinasesof the invention and the AMPK family of protein kinases. Moreover, theresults herein presented indicate that SNARK activity is similar infunctional terms to the activities ascribed to other members of the AMPKfamily. Thus, the expression of SNARK is closely associated with fuelutilization and glucose metabolism and modulation thereof will be usefulto control various cellular responses to endogenous levels of glucoseand other fuels. Moreover, the influence of SNARK on the levels andactivation states of ATP, and the cellular cascades influenced by thoselevels, suggests that SNARK may also have an indirect influence onvarious receptor-based cascades that are driven by ATP Therefore, indiseases, disorders and conditions resulting from aberrant expression orfunction of SNARK, it may be desirable either to increase or decreasethe availability of SNARK endogenously, either by manipulating itsexpression or activity levels or by manipulating the endogenous proteinlevels, using the techniques and agents described hereinabove. Forinstance, it is contemplated that upregulation of SNARK will stimulateliver CPT-1, and thereby enhance lipid metabolism in liver cells and inother cell types such as heart and skeletal muscle. Similarly,activation of SNARK in muscle cells is predicted to increase GLUT-4 andglycogen in muscle. These effects will be similar to those observed whenmuscle cells are treated with insulin. Hence, activation of SNARK ispredicted to have insulin-like effects that would enhance the disposalof glucose into muscle, and thereby reduce plasma glucose, a desirableeffect for the treatment of diabetes and some types of disorders oflipoprotein production leading to increased levels of cholesterol ortriglycerides. In general, it is anticipated that SNARK will be usefulto channel those effects seen to date following administration of AICARto cells, which include increased production of GLUT-4, hexokinase andmuscle glycogen (see for instance Holmes et al, Am. J. Physiol., 1999,1990-1995 and Winder et al, J. App. Physiol., 2000, 88:2219-2226). SNARKtherefore has implications for various disorders involving aberrant fuelutilization and response to metabolic or environmental stress.

[0082] It is contemplated further that SNARK will also influence theresponse from certain cAMP-gated receptors including ion channels, suchas the cAMP-gated Chloride channels, and including the cystic fibrosistransmembrane conductance regulator (CFTR). In particular, it iscontemplated that SNARK participates in this pathway, and may be usefultherapeutically in the treatment of cystic fibrosis by inhibiting thehyper-functioning of the CFTR, as has been contemplated for the AMPKproteins (see Hallows, J. Clin. Invest., 2000, 105(12):1711-1721.

[0083] In one embodiment, SNARK or a variant, chimeric or fragmentthereof may be administered to a subject to prevent or treat a diseaseassociated with decreased expression of SNARK. In another embodiment, anagonist which is specific for SNARK may be administered to a subject toprevent or treat diseases including, but not limited to, those diseaseslisted above. In another further embodiment, a vector capable ofexpressing SNARK, or a fragment or a derivative thereof, may beadministered to a subject to prevent or treat diseases including, butnot limited to, those diseases listed above.

[0084] In a further embodiment, antagonists which decrease theexpression and activity of SNARK may be administered to a subject toprevent or treat diseases predicted to be associated with increasedexpression of SNARK. For example disorders characterized by excessglucose utilization, increased glucose uptake, or decreased glucoseproduction may result in hypoglycemia. In one aspect of the invention aSNARK antagonist may be administered to increase fuel production,decrease glucose uptake, and increase the levels of blood glucose in apatient suffering from hypoglycemia.

[0085] In one aspect, antibodies which specifically bind SNARK may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress SNARK.

[0086] In another embodiment, a vector expressing the complement of thepolynucleotide encoding SNARK may be administered to a subject to treator prevent diseases including, but not limited to, those diseases listedabove.

[0087] In one aspect, antibodies which specifically bind SNARK may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress SNARK.

[0088] In a further embodiment, SNARK or a variant, chimeric or fragmentthereof may be added to cells to stimulate activation of theSNARK-mediated signaling cascade, for instance to drive glucosemetabolism. In particular, SNARK may be added to a cell in culture orcells in vivo using delivery mechanisms such as liposomes, viral basedvectors, or electroinjection for the purpose of promoting cellproliferation and tissue or organ regeneration. Specifically, SNARK maybe added to a cell, cell line, tissue or organ culture in vitro or exvivo to stimulate cell proliferation for use in heterologous orautologous transplantation.

[0089] In an aspect of the present invention, there is provided a methodfor assaying SNARK activity, in which a candidate SNARK protein isincubated with a SNARK substrate under phosphorylating conditions, andthen the extent of phosphorylation is measured. The candidate SNARKprotein is confirmed as having SNARK activity if phosphorylation isdetected in the rank order of substrate selectivity presented in Table 1infra. Similarly, the assay can be exploited to screen and identifycandidate modulators of SNARK activity, by incubating the candidatemodulator with both a SNARK protein and a SNARK substrate underphosphorylating conditions, and then determining whether the candidatemodulator has altered the phosphorylation relative to a controlincubation from which the candidate modulator is absent. Agonists ofSNARK activity are identified by an increase in phosphorylation, whereasantagonists are identified by a decrease in phosphorylation, relative tothe control incubation. In this method, suitable SNARK substratesinclude most substrates known to be phosphorylated by the related AMPKproteins, such as the SAMS peptide identified herein. The assay can beperformed against libraries of small molecules, peptides including SNARKfragments and antibodies, carbohydrates and the like.

[0090] In other embodiments SNARK activators or inhibitors can beexpressed in specific cells and tissues, following which Gene Chip andProteomics Techniques can be used to identify downstream targets in theSNARK signaling pathway that are subsequently amenable for furthermanipulation. Hence, the present invention further provides a method fordefining one or more previously identified or novel genes and proteinsthat may serve as mediators, activators or inactivators of SNARKactivity in cells and tissues.

[0091] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, complementary sequences or vectors ofthe invention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0092] Antagonists or inhibitors of SNARK may be produced using methodsthat are generally known in the art. In particular, purified SNARK maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind SNARK. Antagonists orinhibitors can further be identified as molecules that inhibit thephosphorylation of the SNARK protein, or as molecules that stimulate thedephosphorylation of the SNARK protein. SNARK antagonists may includeSNARK variants in which the functional kinase domains, shown in FIG. 2for rat SNARK for instance, are disrupted by site specific amino acidalteration, to generate inactive SNARK variants that compete withendogenous and functional SNARK for substrate binding. Alternatively,antagonists may be identified as molecules that bind to the SNARKprotein, thereby preventing its functional activation required to exertits cellular effects. Such antagonists of SNARK activity may furtherinclude peptide fragments of SNARK that lack SNARK activity but competewith SNARK for its substrates. Such antagonist fragments may beidentified for instance by deletional analysis of SNARK to truncate oneor both termini, or by cleaving SNARK for instance tryptically orotherwise to generate fragments that can then be examined in thephosphorylation assay to identify antagonists, and also to identifyagonists where desired.

[0093] In another embodiment of the invention, the polynucleotidesencoding SNARK, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding SNARK may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding SNARK. Thus, complementary molecules orfragments may be used to modulate SNARK activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding SNARK.

[0094] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods that are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingSNARK. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0095] Genes encoding SNARK can be turned off by transforming a cell ortissue with expression vectors that express high levels of apolynucleotide or fragment thereof which encodes SNARK. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0096] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding SNARK (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0097] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding SNARK.

[0098] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0099] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding SNARK. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues. RNA molecules may be modified to increaseintracellular stability and half-life. Possible modifications include,but are not limited to, the addition of flanking sequences at the S′and/or 3′ ends of the molecule or the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule. This concept is inherent in the production of PNAs and canbe extended in all of these molecules by the inclusion of nontraditionalbases such as inosine, queosine, and wybutosine, as well as acetyl-,methyl-, thio-, and similarly modified forms of adenine, cytidine,guanine, thymine, and uridine which are not as easily recognized byendogenous endonucleases.

[0100] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0101] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch pets, poultry, livestock, primates, and most preferably, humans.

[0102] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of SNARK,antibodies to SNARK, mimetics, agonists, antagonists, or inhibitors ofSNARK. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0103] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0104] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries that facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0105] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

[0106] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets forinstance. Suitable excipients are carbohydrate or protein fillers, suchas sugars, including lactose, sucrose, mannitol, or sorbitol; starchfrom corn, wheat, rice, potato, or other plants; cellulose, such asmethyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0107] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0108] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is known in the art, e.g., by means of conventional mixing,dissolving, granulating, emulsifying, encapsulating, entrapping, orlyophilizing processes.

[0109] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined buffer prior to use.

[0110] For any compound, the therapeutically effective dose ofSNARK-active compound can be estimated initially either in cell cultureassays, e.g., of neoplastic cells, or in animal models, usually mice,rabbits, dogs, or pigs. The animal model may also be used to determinethe appropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

[0111] A therapeutically effective dose refers to that amount of activeingredient, for example SNARK or fragments thereof, antibodies of SNARK,agonists, antagonists or inhibitors of SNARK, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0112] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors that may be taken intoaccount include the severity of the disease state, general health of thesubject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0113] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0114] In another aspect, antibodies which specifically bind SNARK maybe used for the diagnosis of conditions or diseases characterized byexpression of SNARK, or in assays to monitor patients being treated withSNARK, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for SNARK includemethods which utilize the antibody and a label to detect SNARK in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0115] A variety of protocols including ELISA, RIA, and FACS formeasuring SNARK are known in the art and provide a basis for diagnosingaltered or abnormal levels of SNARK expression. Normal or standardvalues for SNARK expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to SNARK under conditions suitable for complex formationThe amount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of SNARKexpressed in control and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0116] In another embodiment of the invention, the polynucleotidesencoding SNARK may be used for diagnostic purposes. The polynucleotidesthat may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof SNARK may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofSNARK, and to monitor regulation of SNARK levels during therapeuticintervention.

[0117] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding SNARK or closely related molecules, may be used to identifynucleic acid sequences which encode SNARK. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, or the region coding for theC-terminal 320 amino acids of SNARK, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding SNARK, alleles, or related sequences. Alternatively,and particularly for human diagnostics, the probe may have a sequencecapable of revealing the presence of a polynucleotide having all or adetectable portion of any one of the human sequences depicted in FIG.10, or of revealing the complement thereof.

[0118] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the SNARK encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID No: 2, its complement or RNA equivalents thereof, or from genomicsequence including promoter, enhancer elements, and introns of thenaturally occurring SNARK. Useful such sequences are illustrated in FIG.10 for detecting corresponding human DNA. In embodiments of theinvention, such probes are suitably based on the region spanning nucleicacid residues 1-1000 of SEQ ID NO. 2. In the alternative, the probe isbased on the region coding for the C-terminal 320 amino acids of ratSNARK.

[0119] Means for producing specific hybridization probes for DNAsencoding SNARK include the cloning of nucleic acid sequences encodingSNARK or SNARK derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0120] Polynucleotide sequences encoding SNARK may be used for thediagnosis of conditions, disorders, or diseases which are associatedwith either increased or decreased expression of SNARK. Examples of suchconditions or diseases include those associated with fuel utilization,and particularly glucose metabolism, including diabetes, as well asthose associated with aberrant function of cAMP-driven channelsincluding cystic fibrosis. The polynucleotide sequences encoding SNARKmay be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered SNARK expression.

[0121] Such qualitative or quantitative methods are well known in theart.

[0122] In a particular aspect, the nucleotide sequences encoding SNARKand its fragments may be useful in assays that detect activation orinduction of various metabolic disorders, particularly those mentionedabove. The nucleotide sequences encoding SNARK may be labeled bystandard methods, and added to a fluid or tissue sample from a patientunder conditions suitable for the formation of hybridization complexes.After a suitable incubation period, the sample is washed and the signalis quantitated and compared with a standard value. If the amount ofsignal in the biopsied or extracted sample is significantly altered fromthat of a comparable control sample, the nucleotide sequences havehybridized with nucleotide sequences in the sample, and the presence ofaltered levels of nucleotide sequences encoding SNARK in the sampleindicates the presence of the associated disease. Such assays may alsobe used to evaluate the efficacy of a particular therapeutic treatmentregimen in animal studies, in clinical trials, or in monitoring thetreatment of an individual patient.

[0123] In order to provide a basis for the diagnosis of diseaseassociated with expression of SNARK, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes SNARK,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0124] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0125] With respect to diabetes, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. Similarly, detection of an aberrant SNARK gene, byhybridization with a SNARK-encoding polynucleotide or with a probespecific for a region suspected of carrying a mutation, can be used toidentify patients with a genetic anomaly in the SNARK gene. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the condition.

[0126] Additional diagnostic uses for oligonucleotides designed from thesequences encoding SNARK may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′->3′) and another with antisense (3′<-5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0127] Methods which may also be used to quantitate the expression ofSNARK include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 212:229-236). The speed of quantitation of multiple samples maybe accelerated by running the assay in an ELISA format where theoligomer of interest is presented in various dilutions and aspectrophotometric or colorimetric response gives rapid quantitation.

[0128] In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as targets in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information may be used to determine gene function, understandingthe genetic basis of disease, diagnosing disease, and in developing andin monitoring the activities of therapeutic agents.

[0129] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0130] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20 nucleotides in length. For a certain type of microarray, it maybe preferable to use oligonucleotides that are only 7-10 nucleotides inlength. The microarray may contain oligonucleotides that cover the known5′, or 3′, sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray. The “pairs” will be identical, except for one nucleotidewhich preferably is located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from 2 to one million.

[0131] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

[0132] In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devices (slot blot or dot blotapparatus), materials and machines (including robotic instruments) andmay contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any othermultiple from 2 to one million which lends itself to the efficient useof commercially available instrumentation.

[0133] In order to conduct sample analysis using the microarrays, theRNA or DNA from a biological sample is made into hybridization probes.The mRNA is isolated, and cDNA is produced and used as a template tomake antisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or functional analysis of the sequences, mutations, variants, orpolymorphisms among samples (Heller, R. A. et al., (1997) Proc. Natl.Acad. Sci. 94:2150-55).

[0134] In another embodiment of the invention, the nucleic acidsequences that encode SNARK may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0135] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding SNARK on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0136] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0137] In another embodiment of the invention, SNARK, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenSNARK and the agent being tested, may be measured.

[0138] Another technique for drug screening that may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication W084/03564. In this method, as applied to SNARK largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with SNARK or fragments thereof, and washed. BoundSNARK is then detected by methods well known in the art. Purified SNARKcan also be coated directly onto plates for use in the aforementioneddrug screening techniques. Alternatively, non-neutralizing antibodiescan be used to capture the peptide and immobilize it on a solid support.

[0139] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding SNARKspecifically compete with a test compound for binding SNARK. In thismanner, the antibodies can be used to detect the presence of any peptidethat shares one or more antigenic determinants with SNARK.

EXAMPLES Materials and Methods

[0140] Chemicals and Materials—Radiochemicals were purchased from eitherICN Biomedicals (California, USA; [³²P]-α-dATP (>3000 Ci/mmol)),Amersham Pharmacia Biotech (Baie D'Urfé, Quebec; [³²P]-γ-ATP(>5000Ci/mmol)) or from NEN Life Sciences (Guelph, Canada; [³⁵S]-methionine(>1000 Ci/mmol)). Cell culture supplies, the Concert nucleic acidpurification and Super Script preamplification systems were obtainedfrom Canadian Life Technologies (Burlington, Ontario). The TOPO TAcloning kit and pcDNA3.1 vector were purchased from Invitrogen (SanDiego, Calif.). Nylon and PVDF membranes, the T7-Sequencing kit, the GSTgene fusion system and protein A Sepharose CL4B were from AmershamPharmacia Biotech. The TNT coupled reticulocyte lysate system was fromPromega (Madison, Wis.). The Bradford DC Protein assay kit was purchasedfrom BioRad (Mississauga, Canada). The NEN Renaissance enhancedchemiluminescence (ECL) reagent plus kit and the Kodak BioMax MS and MLfilms were purchased from Mandel Scientific (Guelph, Ontario). Themajority of chemicals and protease inhibitors were purchased fromBioShop (Oakville, Ontario). Dephosphorylated myelin basic protein (MBP)was obtained from Upstate Biotechnology (New York, N.Y., U.S.A.).Dephosphorylated B-casein, whole histone and protamine sulphate werepuchased from Sigma-Aldrich (Oakville, Ontario, Canada). Protein kinaseC (PKC) and cAMP-dependent protein kinase (PKA) inhibitor peptides weresupplied by Santa Cruz Biotechnology (Santa Cruz, Calif., U.S.A.). TheSAMS peptide was synthesized by the Protein Synthesis Facility (Hospitalfor Sick Children, Toronto, Ontario, Canada). AMPKa2 antibody was kindlyprovided by Dr. Neil Ruderman (Boston, Mass., U.S.A.).

[0141] Cell Culture and Irradiation—Neonatal rat keratinocytes (NRKC)and Baby hamster kidney (BHK) cells were propagated as describedpreviously [21,22]. Cells were seeded into 10 cm dishes at a density of1×10⁶ NRKC cells or 3×10⁶ BHK cells and were incubated for 2-3 daysprior to experimentation. The medium was removed and replaced withlow-serum medium, i.e., DMEM containing 0.1% newborn calf serum (CS),100 μg/ml streptomycin and 100 units/ml penicillin for and incubated foreither 18 hours (BHK cells) or 1.5 hours (NRKC cells) prior to eachexperiment. Glucose deprivation was performed as previously described[10], except that 25 mM glucose was used for control plates.

[0142] DNA and RNA analysis—Sequencing of SNARK cDNAs was performed withSp6 and T7 primers using the Sequenase T7 DNA Polymerase kit or by theYork University Core Sequencing Facility (Toronto, Canada) using anApplied Biosystems Sequencer-Stretch Model and the Taq Polymerase DyeDioxy terminator cycle sequencing method. Clustal multiple sequencealignment was performed using the MBS-Aligner program. RNA was isolatedand analyzed by Northern blotting and RT-PCR as previously described[23]. The full-length SNARK CDNA was labelled with [α-P³²]-ATP by randompriming technique [24] and used as a probe for Northern and Southernanalyses as previously described [23].

[0143] Reverse Transcriptase-PCR—First-strand cDNA was generated usingthe SuperScript preamplification system and the following primers;5′-CCGGATCCATGGAGTCGGTG-GCCTTACAC-3′ and5′-CCGGATCCCTAAGAGTTCCCCAG-ACTCA-3′ (SNARK sequences bolded) to amplifySNARK transcripts. PCR was performed with the Perkin Elmer GeneAmp 2400PCR system using Pfu DNA polymerase in a final volume of 50 μl. Thefollowing conditions were used: denaturation at 94° C. for 5 min, 35cycles consisting of 94° C. for 1 min, 59° C. for 1 min and 72° C. for 2min, and final extension at 72° C. for 10 min. Half of the PCR reactionwas loaded on a 1% agarose gel and immobilized on a nylon membrane andthen probed as described above. PCR products were subcloned using theTOPO TA Cloning kit and sequenced to verify identity of SNARK PCRproducts.

[0144] Human Chromosomal Mapping Following isolation of a partial humanSNARK genomic fragment, chromosomal localization was performed byfluorescence in situ hybridization (FISH)[25] to normal human lymphocytechromosomes counterstained with propidium iodide and4′,6-diamidin-2-phenylindol-dihydrochloride (DAPI).

[0145] Cloning of SNARK cDNAs—The full-length clone containing theentire open reading frame of SNARK was generated using overlappingclones isolated from rat lung, kidney and keratinocytes. The completeopen reading frame of rat SNARK was subcloned into the pcDNA3.1 vector.

[0146] Cloning of human SNARK was completed as follows: The humankeratinocyte cell line, HaCaT, is an immortalized epithelial cell linefrom adult human skin that exhibits a transformed phenotype, but remainsnontumorigenic (Boukamp P, et al. 1988 J Cell Biol 106:761-7). Totalcellular RNA was isolated from HaCaT cells, as previously described(Chirgwin J M, et al., 1979 Biochemistry 18:5294-99) and first-strandcDNA was generated using the SuperScript preamplification system(Canadian Life Technologies; Burlington, Ontario). Reversetranscriptase-polymerase chain reaction (RT-PCR) was performed usingHaCaT first-strand cDNA, a SNARK sense-strand primer (nt 240-260;5′-tgaggcaccgctacgagttcc-3′) and anti-sense strand primer (nt 918-938;5′-accggatcaggccacaggcat-3′) in order to amplify a 698 bp region of theSNARK transcript. RT-PCR was performed with the Perkin Elmer GeneAmp2400 PCR system using Taq DNA polymerase (Canadian Life Technologies;Burlington, Ontario) in a final volume of 50 μl. The followingconditions were used: denaturation at 94° C. for 5 min, 35 cyclesconsisting of 94° C. for 1 min, 59° C. for 1 min and 72° C. for 2 min,and final extension at 72° C. for 10 min. The resulting PCR reactionproduct was analyzed by size separation on a 1% agarose gel. The 698 bpband was excised from the agarose and the DNA was eluted using theConcert nucleic acid purification system (Canadian Life Technologies;Burlington, Ontario). PCR products were subcloned using the TOPO TACloning kit (Invitrogen; San Diego, Calif.) and sequencing was performedby ACGT Sequencing (Toronto, Ontario) with T7 and M13 primers. Sequencealignment and comparison of HaCaT and rat SNARK sequences was performedusing the DNASIS program (Hitachi Software Engineering).

[0147] Cloning of mouse SNARK was completed as follows: Hairless micewere euthanized and kidneys were dissected and processed for RNAextraction (Chirgwin J M, et al., 1979 Biochemistry 18:5294-99). Totalcellular RNA was isolated and first-strand cDNA was generated using theSuperScript preamplification system (Canadian Life Technologies;Burlington, Ontario). Reverse transcriptase-polymerase chain reaction(RT-PCR) was performed using mouse kidney first-strand cDNA, a SNARKsense-strand primer (nt 240-260; 5′-tgaggcaccgctacgagttcc-3′) and eitherof the anti-sense strand primers, 1) 5′-accggatcaggccacaggcat-3′(nt918-938), or 2) 5′-ccagttgacccaccaatgactgg-3′(nt 987-1001) in order toamplify either, a 1) 698 bp, or 2) 761 bp region of the SNARKtranscript. RT-PCR was performed with the Perkin Elmer GeneAmp 2400 PCRsystem using 2.5 Units of Pfu DNA polymerase (Stratagene, Calif., USA)in a final volume of 50 μl. The following conditions were used:denaturation at 94° C. for 5 min, 30 cycles consisting of 94° C. for 1min, 53° C. for 1 min and 72° C. for 2 min, and final extension at 72°C. for 20 min. The resulting PCR reaction product was analyzed by sizeseparation on a 1% agarose gel. The PCR products were excised from theagarose and the DNA was eluted using the Concert nucleic acidpurification system (Canadian Life Technologies; Burlington, Ontario).PCR products were subcloned using the TOPO TA Cloning kit (Invitrogen;San Diego, Calif.) and sequencing was performed by ACGT Sequencing(Toronto, Ontario) with T7 and M13 primers. Sequence alignment andcomparison of HaCaT and rat SNARK sequences was performed using theDNASIS program (Hitachi Software Engineering).

[0148] Generation of Antisera—A polypeptide containing 203 residues(corresponding to nucleotides 858-1467 of SEQ ID NO. 2) was expressed asa glutathione S-transferase (GST) fusion protein, purified bypolyacrylamide electrophoresis and used to immunize three rabbits(Cocalico Biologicals, USA). Polyclonal anti-SNARK antiserum wascollected and used for Western blot analysis and immunoprecipitations.

[0149] In vitro Transcription and Translation—The SNARK/pcDNA3.1 plasmidconstruct was used as the template for in vitro transcription andtranslation. The TNT coupled reticulocyte lysate system was usedaccording to the manufacturer's protocol. Immunoprecipitation (IP) [29]of ³⁵S-SNARK and 35S-luciferase (system control) was performed with 2 μlof the TNT reactions as described below. Twenty μl of 2X SDS-loadingbuffer (250 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, 0.04%(w/v) bromophenol blue) was added to the complexes prior toelectrophoresis on a 10% polyacrylamide/SDS gel. The gel was dried andthen exposed to Kodak BioMax MS film.

[0150] Protein extracts, Immunoprecipitations and Western Analysis—Cellcultures were scraped and homogenized in lysis buffer as previouslydescribed [27]. The total protein concentration was assayed by BradfordDC protein assay. Immunoprecipitation was performed with 500 μg of totalprotein as previously described [10], except that the finalconcentration of NaCl in the IP reaction was 150 mM. Western analysis ofimmune complexes samples was carried out by electrophoresis on an 8%polyacrylamide/SDS gel followed by transfer and immobilization ofproteins on a PVDF membrane. Membranes were blocked with 2% gelatin inTris-buffered saline (0.5 M Tris, 1.5 M NaCl) with 0.1% Tween-20 (TBST)for 2 hours, incubated with SNARK antiserum (1:1600 dilution in TBST)for 4 hours and then incubation with a secondary antibody of horseradishperoxidase-linked anti-rabbit IgG (Amersham Pharmacia Biotech) for 1hour. After extensive washing, the membrane was developed with ECL for60 seconds and exposed to Kodak BioMax ML film. Equal loading of proteinwas verified by staining with Ponceau S.

[0151] Kinase Assay—Following immunoprecipitation with antiserum, SNARKactivity was analysed by performing kinase assays withimmunoprecipitated SNARK and a variety of substrates (MBP, B-casein,whole histone fraction, protamine sulphate and a synthetic peptidesubstrate (HMRSAMSGLHLVKRR, ‘SAMS’ peptide). Kinase assays with the SAMSpeptide were performed essentially as described [28,29], except that thethree washes were done with IP buffer containing 500 mM NaCl followed bya final wash in kinase assay buffer. For kinase assays performed todetermine substrate specificity (with substrates other than SAMSpeptide), reactions included 30 ug of substrate, 500 nM PKA inhibitorpeptide and 1 uM PKC inhibitor peptide. Activities were calculated asfmol of phosphate incorporated into the SAMS peptide/min per milligramof lysate subjected to immunoprecipitation, minus the activity obtainedwith a blank reaction (cell lysate and Protein A-Sepharose only).

[0152] Cell Transfections—Stable SNARK-transfected cell lines weregenerated using the calcium phosphate-mediated method of DNA transfer asdescribed previously using G418 to select for successful transfectants[30].

[0153] Results

[0154] Southern blot analysis demonstrated a simple pattern ofhybridizing bands in both rat and human genomic DNA samples, consistentwith a single copy of SNARK in the mammalian genome (FIG. 1A). The humanchromosomal localization of SNARK was examined by hybridizing anisolated rat SNARK cDNA fragment with a human P1-derived artificialchromosome (PAC) library. This experiment identified a singlehybridizing genomic PAC clone that localized the human SNARK homologueto human chromosome 1q32 (FIG. 1B). Positive hybridization signals at1q32 were noted in >90% of the metaphasic cells.

[0155] Using the original partial cDNA [20] as a probe, a full-lengthcDNA clone containing 2929 nucleotides was isolated with a single,uninterrupted ORF of 1893 nucleotides, beginning at nucleotide 83 andterminating at position 1975 (FIG. 2). The ORF encoded a putativeprotein of 630 amino acids (aa) with a predicted molecular mass of 69.95kiloDaltons (kD) and a theoretical pI of 9.35. Comparison of the deducedamino acid sequence of the amino (N)-terminal region of the protein (aa57-308) with other known proteins revealed 48% and 50% identity (68%similarity by including conservative substitutions scored by theBLOSUM62 matrix) within the catalytic domain of SNF1 protein kinase [31]and AMPK [32], respectively, prompting the designation ofSNF1/AMPK-Related Kinase, or SNARK (FIG. 3). SNARK contains all 11catalytic subdomains conserved in serine/threonine protein kinases [33](FIG. 3) Analysis of the catalytic domain of SNARK using the Prositeprogram revealed a protein kinase ATP-binding region signature (aa63-89) and a serine/threonine protein kinase active-site signature (aa175-187) (FIG. 2).). The sequences at the carboxyl (C)-terminus of SNARKwere distinct and not well conserved with C-terminal sequences of otherSNF1/AMPK family members. The instability index was computed to be 58.40using the Protparam Tool program, classifying SNARK as an unstableprotein.

[0156] Northern analysis demonstrated SNARK RNA transcripts were mostabundant in rat kidney (FIG. 4A). RT-PCR detected two SNARK CDNAproducts in RNA from rat heart, skin, spleen, lung, uterus, liver and aneonatal rat keratinocyte cell line (FIG. 4B). The two different SNARKRT-PCR products were cloned from several tissues, sequenced and werefound to encode either authentic SNARK (1437 bp) or aninternally-deleted SNARK (-Δ) transcript (1247 bp). While rat kidneycontained predominantly the intact SNARK transcript and testes expressedonly the 1247 bp SNARK-Δ transcript, both intact and Δ-SNARK transcriptswere detected in skin, spleen, lung, uterus and liver. The SNARK-Δtranscript contained a 57 bp in-frame deletion, spanning parts of kinasedomains I and II, and a 133 bp out-of-frame deletion in kinase domainsIX-XI, including the invariant lysine residue involved in maximal enzymeactivity. Translation of the SNARK-Δ transcript is predicted to giverise to a prematurely terminated protein of ˜415 amino acids. Internallydeleted rat AMPK transcripts have also been reported [347]. The probesof the present invention, based on SNARK-encoding DNA of SEQ ID NO. 2,are thus useful to identify aberrant SNARK-encoding DNA in tissuesamples, and can be used diagnostically to characterize DNA samplesobtained from patents presenting with disorders related to aberrantglucose metabolism.

[0157] In vitro transcription and translation using the full-lengthSNARK cDNA template in the presence of [³⁵S]-methionine, resulted in amajor protein product of ≈76 kD (FIG. 5A, lane 1) that wasimmunoprecipitated by SNARK antiserum (lane 5). A clearly detectableprotein doublet, with a size of approximately 76-80 kD, was detected intwo separate clones of SNARK-transfected BHK cells (BHK+1 and BHK+11)using SNARK antiserum (FIG. 5B; lanes 1 and 3), but not with non-immuneserum (FIG. 5B; lane 2).

[0158] To assess whether SNARK was capable of autophosphorylation,immunoprecipitated SNARK was incubated with [³²p]-γ-ATP and reactionproducts were examined by SDS-Polyacrylamide gel electrophoresis (FIG.6). Although, no autophosphorylated products were detected in samples ofimmunoprecipitated endogenous SNARK from wildtype BHK cells (lane 1) ,one major phosphorylated band, possibly a protein doublet, was detectedin the immunoprecipitates from SNARK-transfected BHK cells (FIG. 6;lanes 2 and 4). The size of the phosphorylated band(s) corresponds tothe size of SNARK detected in these cell lines by Western analysis(≈76-80 kD). Furthermore, no phosphorylated proteins were observed incell extracts following immunoprecipitation with non-immune serum (lanes3 and 5) or samples containing extract but no antiserum (lane 6). Theseresults indicate that SNARK is a protein kinase capable ofautophosphorylation in vitro. This assay is also suitable fordetermining whether SNARK variants retain the autophosphorylatingproperties of SNARK.

[0159] To determine whether immunoprecipitated SNARK protein possessedthe ability to phosphorylate protein substrates in vitro, kinase assayswere performed using candidate substrates including dephosphorylatedMBP, dephosphorylated β-casein, whole histone fraction, protaminesulfate, and the SAMS peptide, a well-established AMPK substratecorresponding to the site in rat acetyl-CoA carboxylase phosphorylatedby AMPK [28]. Peptide inhibitors of PKA and PKC were included in thesereactions to eliminate phosphorylation of these substrates by theseenzymes. In the assays performed with SNARK immunoprecipitated from NRKCcell lysates, SNARK was able to phosphorylate SAMS peptide, but itsability to phosphorylate MBP, β-casein, whole histone fraction andprotamine sulfate was minimal. The ability of SNARK to phosphorylate theSAMS peptide substrate was unaffected by the presence of PKA and PKCinhibitors, indicating that the observed kinase activity was not due tothe phosphorylation of SAMS peptide by PKA or PKC. Results are presentedin the Table below: TABLE 1 Substrate Specificity of the SNARK proteinActivity Substrate (fmol/min per mg) SAMS peptide 145 ± 46* MBP 91 ± 11B-casein 37 ± 12 Whole histone fraction 19 ± 6  Protamine sulfate 7 ± 2

[0160] Immunoprecipitation of 500 ug of NRKC cell lysate with SNARKantiserum was performed as described above. Phosphotransferase activitywas assayed with either SAMS peptide (250 uM) or 30 ug ofdephosphorylated MBP, dephosphorylated β-casein, whole histone fractionor protamine sulfate in the presence of 1 μM PKC inhibitor and 500 nMPKA inhibitor peptides. Phosphortransferase activity is expressed infmol of phosphate transferred to the SAMS peptide/min at 30 C per mg ofprotein subjected to immunoprecipitation. Results are means±S.E.M. fortwo individual assays with at least eight samples per group. *p<0.01compared with all other substrates.

[0161] Kinase assays performed with the SAMS peptide onimmunoprecipitated SNARK from wildtype BHK cells gave a low basal levelof SAMS phosphotransferase activity (FIG. 7A, solid box). In contrast,the basal SNARK phosphotransferase activity detected inSNARK-transfected BHK cells (BHK+1) and in rat NRKC cells was 3.4-foldand 2-fold higher, respectively, than the levels found in wildtype BHKcells (FIG. 7, solid boxes), Since AMPK-α2 antibodies immunoprecipitate4-fold less SAMS phosphotransferase activity than the SNARK antiserum inSNARK-transfected BHK cells (data not shown), the phosphotransferaseactivity detected in this assay system appears to be specific for SNARKkinase activity. The SNARK antiserum does not cross-react with AMPKisoforms in rat skeletal muscle and AMPK-α2 antibodies do notimmunoprecipitate any detectable SAMS phosphotransferase activity inSNARK-transfected BHK cells, these findings indicate that SNARK exhibitsAMPK-like kinase activity.

[0162] The kinase assay just described is useful to identify functionalvariants of SNARK, and chimeric forms of SNARK, that retain itsphosphorylation properties. The kinase assay is also useful to identifysuch variants and chimerics of SNARK that retain its substrate activityin hierarchal terms relative to the substrates tested. That is, it isexpected that all SNARK proteins, whether wildtype (such as rat SNARKand its mammalian homologs), variant, or chimeric, will exhibit the rankorder of phosphorylating activity shown in Table 1 above with respect tothose kinase substrates.

[0163] AMPK is activated by environmental stresses that lead todepletion of cellular ATP and elevation of AMP [1]. To evaluate theeffects of cellular stress on SNARK activity, there was examined theeffects of AMP on SNARK phosphotransferase activity in wildtype BHK,SNARK-transfected BHK and NRKC cell lines. Although no significantchange in SNARK phosphotransferase activity was observed when wildtypeand SNARK-transfected cell lysates were assayed in the presence of 200μM AMP, SNARK phosphotransferase activity increased by 1.7 fold(p<0.001) in NRKC cells (FIG. 7, hatched box).

[0164] The adenosine analogue, 5-aminoimidizole-4-carboxamide riboside(AICAR), provides a means of stimulating AMPK activity in whole cells,even in the presence of high glucose concentrations, and mimics theeffects of AMP on the AMPK cascade [35,36]. Intriguingly, SNARK activitywas induced 2.8-fold (p<0.05) in NRKC cells when treated with 1 mM AICARfor one hour (FIG. 8A, hatched box), as compared to SNARK activitymeasured in untreated NRKC cells (solid box). Since AICAR is taken upinto cells and converted by adenosine kinase into a phosphorylatedmonophosphate form (ZMP) which mimics the effects of AMP on both theallosteric activation and the phosphorylation of AMPK via AMPK kinase,this data implies that SNARK can be covalently modified by AMPK kinasein response to elevated cellular AMP.

[0165] The concentration of glucose in culture medium is an importantmodulator of both SNF1 activity in yeast cells [37, 38] and AMPKactivity in pancreatic β-cells [10]. SNARK activity assayed in wildtypeBHK cells deprived of glucose for 90 minutes was 2.6-fold higher(p<0.03; FIG. 8B, hatched box) than activity levels measured in BHKcells cultured in 25 mM glucose (solid box). This result suggests thatSNARK activity responds to glucose deprivation in a manner similar toyeast SNF1 and rat AMPK [10, 38]. Furthermore, immunoreactive SNARK waslocalized to the exocrine and endocrine compartments of the humanpancreas. Consistent with this finding was the Western analysis of celllysates from the rat INS-1 (insulinoma), mouse αTC (glucagonoma) andhamster InR1G9 (glucagonoma) cell lines which revealed SNARKimmunoreactive proteins in the INS-1 and αTC cell lines corresponding insize to that found in SNARK-transfected BHK cells (i.e., approx 80 kDa).Although no 80 kDa SNARK protein was detected in the hamster cell lineInR1G9, a protein migrating in approximately 106 kDa was detected inboth the InR1G9 and SNARK-transfected BHK cells. This larger proteinmight represent a form of SNARK that undergoes differentialpost-translational modification.

[0166] This study describes the cloning of a member of the SNF1/AMPKfamily of serine/threonine kinases localized to human chromosome 1q32.The 3.5 kb SNARK mRNA encodes for a 76-80 kDa protein containing aminoacid motifs characteristic of serine/threonine kinases. SNARK mRNAtranscripts were detectable by RT-PCR in almost all tissues examined,hence like AMPK [28, 34, SNARK is not a cell-specific kinase. Thedetection of two SNARK RNA isoforms, including the SNARK (-Δ) transcriptthat is predicted to give rise to a non-kinase protein, highlights theimportance of using probes or primers specific for detection offull-length SNARK in future studies of SNARK expression and localizationin cell types. Similarly, both AMPK and SNARK are able to phosphorylatethe SAMS peptide substrate, derived from the site on acetyl Co-Acarboxylase that is specifically phosphorylated by AMPK. Importantly,the antisera used in our studies for analysis of SNARKautophosphorylation and kinase activity was directed against peptidesequences in the carboxy-terminal region of SNARK that exhibit nohomology to AMPK. Taken together, the structural and functional dataestablish SNARK as a new mammalian member of the SNF1/AMPK family ofkinases.

[0167] Within its catalytic domain, SNARK is most closely related to theSNF1/AMPK family of protein kinases, possessing a high degree ofhomology at the amino acid level. Members of the SNF1/AMPK proteinkinase family have been highly conserved throughout evolution and thehallmark members of this family, SNF1 and AMPK, are generally thought torepresent key metabolic sensors in stress response systems, althougheach responds to different types of stresses. AMPK is activated byenvironmental and cellular stresses [1], including exercise and glucosedeprivation [10,39]. These stresses deplete cellular ATP and, via theadenylate kinase reaction, elevate AMP which serves as a switch toactivate AMPK activity. The finding that SNARK activity can bestimulated by exposure to both AMP and AICAR suggests that SNARK, likeAMPK signaling cascade, is sensitive to levels of cellular AMP.

[0168] Although UVB is a constant source of cellular stress for the skincell since it is a major component of terrestrial sunlight, themolecular signaling mechanisms induced by UVB are incompletelyunderstood. It has been reported that UVB significantly activates c-JunNH₂-terminal kinases (JNKs) in keratinocytes and induces translocationof membrane-associated protein kinase C isoforms from cytosol tomembrane in epidermal cells mediating signal transduction and apoptosisthrough activation of extracellular-regulated kinases (Erks) and JNKs.In addition, the FKBP/FRAP/p70S6K signaling cascade has been identifiedas a pathway regulated by UVB-induced DNA damage and repair.Furthermore, UVB-induces growth inhibition of keratinocytes inhyperproliferative skin disorders via downregulation of the type IIinterleukin-8 receptor (CXCR-2). These and other studies support thecontention that the UVB signal is transduced via bothmembrane-associated and cytosolic signal pathways to the nucleus,resulting in multiple cutaneous effects. It is conceivable that theseresponses to UVB radiation may lead to ATP:AMP ratio perturbations whichactivate SNARK in keratinocytes in an attempt to re-establish metabolicequilibrium within the cell.

[0169] As noted, SNARK activity can be regulated by the concentration ofglucose in the medium. This is consistent with recent experimentsdemonstrating that AMPK in pancreatic β-cells is modulated in responseto the extracellular glucose concentration [10]. Glucose deprivation ofpancreatic β cell lines resulted in a >5-fold activation of AMPKactivity within 30 minutes of glucose removal [10]. AMPK activation wasassociated with a large increase in the cellular AMP/ATP ratio resultingfrom the low levels of extracellular glucose and correlated inverselywith insulin secretion. Conversely, AMPK activity was inhibited byincreasing glucose concentrations in MIN6 beta cells andimmunoneutralization of the AMPK complex diminished glucose-regulatedgene transcription in vitro [40]. Because SNARK immunoreactivity is alsolocalized to human islets and rodent islet cell lines, it is likely thatSNARK is modulator of islet cell response to metabolic stress, such ashypoglycemia.

[0170] The citations referenced herein are incorporated herein byreference, and are listed below in full:

[0171] 1. Hardie et al, 1998, Ann. Rev. Biochem., 67:821.

[0172] 2. Carlson, 1999, Curr. Opinion Microbiol., 2:202

[0173] 3. Alderson et al, 1991, Proc. Nati. Acad. Sci. USA, 88:8602

[0174] 4. Le Guen et al, 1992, Gene, 120:249

[0175] 5. Bouly et al, 1999, Plant J., 18:541

[0176] 6. Hannappel et al, 1995, Plant Mol. Biol., 27:1235

[0177] 7. Halford et al, 1992, Plant J., 2:791

[0178] 8. Muranaka et al, 1994, Mol. Cell. Biol., 14:2958

[0179] 9. Takano et al, 1998, Mol. Gen. Genet., 260:388

[0180] 10. Salt et al, 1998, Biochem. J., 335:533

[0181] 11. Sugden et al, 1999, Plant Physiol., 120:257

[0182] 12. Bracchi et al, 1996, Mol. Biochem. Parasitol., 76:299

[0183] 13. Davies et al, 1999, Plant Cell, 111: 1179

[0184] 14. Wang et al, 1999, FEBS Lett., 453:135

[0185] 15. Becker et al, 1996, Eur. J. Biochem., 235:736

[0186] 16. Heyer et al, 1997, Mol. Reprod. Dev., 47:148

[0187] 17. Ruiz et al, 1994, Mech. Dev., 48:153

[0188] 18. Inglis et al, 1993, Mamm. Genome, 4:401

[0189] 19. Gardner et al, 2000, Genomics, 63:46

[0190] 20. Rosen et al, 1995, Am. J. Physiol., 268:C846

[0191] 21. Rosen et al, 1990, Cancer Res., 50:2631

[0192] 22. Yusta et al, 1999, J. Biol. Chem., 274:30459

[0193] 23. Shahmolky et al, 1999, Photochem Photobiol., 70:341

[0194] 24. Feinberg & Vogelstein, 1983, Anal. Biochem., 132:6

[0195] 25. Lichter et al, 1990, Science, 247:64

[0196] 26. Ip & Davis, 1998, Curr. Opin. Cell Biol., 10:205

[0197] 27. Salt et al, 1998, Biochem. J., 334:177

[0198] 28. Davies et al, 1989, Eur. J. Biochem., 186:123

[0199] 29. Dale et al, 1995, FEBS Lett., 361:191

[0200] 30. Chen & Okayama, 1988, BioTechniques, 6:632

[0201] 31. Celenza & Carson, 1986, Science, 233:1175

[0202] 32. Carling et al, 1994, J. Biol. Chem., 269:11442

[0203] 33. Hanks & Hunter, 1995, FASEB J., 9:576

[0204] 34. Gao et al, 1995, Biochim. Biophys. Acta., 1266:73

[0205] 35. Corton et al, 1995, Eur. J. Biochem., 229:19509

[0206] 36. Henin et al, 1996, Biochim. Biophys. Acta., 1290:197

[0207] 37. Woods et al, 1994, J. Biol. Chem., 269:19509

[0208] 38. Wilson et al, 1996, Curr. Biol., 6:1426

[0209] 39. Winder & Hardie, 1996, Am. J. Physiol., 270:E299

[0210] 40. da Silva et al, 2000, Proc. Natl. Acad. Sci. USA, 97:4023

1 109 1 2929 DNA RAT 1 ggcacgaggt gacctctgag cctgcggctc tccgcgcgctgctgctgctg cccgaccccc 60 tccgcctcgc cgtccccgca ccatggagtc ggtggccttacaccggcgcg ggaacctggc 120 tccctcggcc tccgccctgg ccacggagag cgcccggccgctggcggacc ggctcatcaa 180 gtcgcccaaa cctctgatga agaagcaggc ggtgaagcggcaccatcaca aacacaacct 240 gaggcaccgc tacgagttcc tggagaccct gggcaagggcacctacggga aggtgaagaa 300 agcacgagag agctcgggac gcctggtggc catcaagtctatcaggaagg acaaaatcaa 360 agatgagcag gatctgttgc acataaggag ggagatcgagatcatgtctt cactcaacca 420 cccccacatc attgccatcc atgaagtgtt tgagaacagcagcaagattg tgattgtcat 480 ggagtacgcc agccgaggcg atctgtacga ttacatcagtgagcggccac ggctgaatga 540 gcgggacgcc aggcatttct tccgacagat cgtgtccgccctgcactact gccaccagaa 600 cgggattgtt caccgggacc tcaagctgga gaacatccttctagatgcca gtggcaacat 660 caagattgct gattttggcc tctccaacct gtatcacaaaggcaagttcc tccagacgtt 720 ctgtgggagc cctctctatg cctcacctga gatcgtcaacgggaagccct atgtgggccc 780 agaggtggac agctggtctc tgggcgttct tctgtacatcctggtgcatg gcaccatgcc 840 ctttgacggg caggatcata aaaccctggt gaaacaaatcagtagcgggg cttaccgaga 900 gccgtgcaaa ccgtctgatg cctgtggcct gatccggtggctgttaatgg tgaatcccat 960 ccgtcgggcc actctggagg atgtagccag tcattggtgggtcaactggg gttacagcac 1020 ccgaattggg gaacaggaag ctctgcgaga gggtgggcaccctagcggtg actctggccg 1080 ggcctctatg gcggactggt tacgtcgctc ctcccgccccctcctggaga atggagccaa 1140 agtgtgtagc ttcttcaagc agcatgtgcc gggaggtggaagcacgggac cggggctgga 1200 gcggcaacat tctcttaaga agtcccgcaa ggagaatgacatggctcaga ctctgcagaa 1260 tgacccagtt gaagatactt cctctcgccc tggcaagaacagcctcaagc ttccgaaagg 1320 tatcctcaag aaaaaggcct ctccctcatc gggggaggtacaggagggcc ctcaggaact 1380 cagaccagtg tccaataccc cagggcagcc tgtccctgctatacccctgc tcccaaggaa 1440 gggcattctt aagaagtctc ggcagcgtga atctggttactactcctctc cagagcccag 1500 tgagtctggg gaactcttag acgcaggtga tgtgtttgtgagtggggacc ccgtggagca 1560 gaagtctcca caagcttcag ggcgcctcca tcgcaagggcatcctcaaac tcaatggcaa 1620 gttttcccgc acagccttag aaggcactgc ccctagcacctttggctccc tagaccaact 1680 ggcctcccct catcctacag cccgggccag ccgtccctcgggagctgtga gtgaggacag 1740 catcctgtcc tccgagtcct ttgaccaatt ggacttgcccgagcggcttc ccgaaacccc 1800 actgaggagc tgtgtgtctg tggacaacct gaggaggcttgagcagcctc cctcagaagg 1860 cctaaaacga tggtggcagg aatccttggg ggatagccgcttttctctga cagactgcca 1920 agaggtgaca gcagcctaca gacaagccct aggaatctgctcgaagctca gctgaggagg 1980 agaggcagtg ccccagtgat ggggtagact cttagaggggtttgcagagg aacctgggta 2040 gattccccag ggttgtagag tacatcaaga actctctctctgtcttcagc ctgattgaac 2100 ctggaggctg agagaaatag cagagatatg gaaaggactgacctacagag tctgactgca 2160 gatgtgagcg gcacagagac tgaaagtgcc tacctcctttatgctgagtg ctacccatgg 2220 catctccccc ctgctctctg ccagtgtcag ggtgtacccacataagttcc tgttcgcatc 2280 gaccaccagg gttagaaccc tgacatccct ggaagtaatgtggagcaacc tcgcttattt 2340 aaagaggaaa cagcctctgg tttccatctc tgctgctgtgcatctcaaag acctagaaag 2400 actcaactgc tgtttcactt catctcaagg ggacctcagagacctgagcc ttgaagctgt 2460 tcctgataac cagactatga tggatatgtc tgtttctcaggccagcagga cccagaatgt 2520 gctgacttat ttatttttgt gattctcact tctggtttctgtttcgtttt tttgttgttg 2580 gttttgttgt tgtcgtttgt ttcttgtttt ttgtttgtttgtttgtttgt ttgtttgttt 2640 tttaaagtga attttgctgc tttcggtaat gtgaatgctgtgttctgggg aaagccactg 2700 tgtcattgaa gtgtgtgtac agagaagtat ttggcagtgattccttctaa tggggggtgg 2760 ccttttcaga tgtatgtctt gagcactgtc tggattgggtctcctgtccc ctcacaccag 2820 aggctgtcca ccctccctca tctgtggcca aaaaaaacctcattaaaacc agcaacggca 2880 actggaaaaa aaaaaatttt tttttttttt ttttttttttttttttttt 2929 2 177 DNA RAT 2 gcccggccgc tggcggaccg gctcatcaagtcgcccaaac ctctgatgaa gaagcaggcg 60 gtgaagcggc accatcacaa acacaacctgaggcaccgct acgagttcct ggagaccctg 120 ggcaagggca cctacgggaa ggtgaagaaagcacgagaga gctcgggacg cctggtg 177 3 122 DNA RAT 3 ggtggccatc aagtctatcaggaaggacaa aatcaaagat gagcaggatc tgttgcacat 60 aaggagggag atcgagatcatgtcttcact caaccacccc cacatcattg ccatccatga 120 ag 122 4 150 DNA RAT 4agtgtttgag aacagcagca agattgtgat tgtcatggag tacgccagcc gaggcgatct 60gtacgattac atcagtgagc ggccacggct gaatgagcgg gacgccaggc atttcttccg 120acagatcgtg tccgccctgc actactgcca 150 5 109 DNA RAT 5 agattgctgattttggcctc tccaacctgt atcacaaagg caagttcctc cagacgttct 60 gtgggagccctctctatgcc tcacctgaga tcgtcaacgg gaagcccta 109 6 11 DNA RAT 6 tgtgggcccag 11 7 121 DNA RAT 7 aggtggacag ctggtctctg ggcgttcttc tgtacatcctggtgcatggc accatgccct 60 ttgacgggca ggatcataaa accctggtga aacaaatcagtagcggggct taccgagagc 120 c 121 8 13 DNA RAT 8 gtgcaaaccg tct 13 9 421DNA RAT 9 gatgcctgtg gcctgatccg gtggctgtta atggtgaatc ccatccgtcgggccactctg 60 gaggatgtag ccagtcattg gtgggtcaac tggggttaca gcacccgaattggggaacag 120 gaagctctgc gagagggtgg gcaccctagc ggtgactctg gccgggcctctatggcggac 180 tggttacgtc gctcctcccg ccccctcctg gagaatggag ccaaagtgtgtagcttcttc 240 aagcagcatg tgccgggagg tggaagcacg ggaccggggc tggagcggcaacattctctt 300 aagaagtccc gcaaggagaa tgacatggct cagactctgc agaatgacccagttgaagat 360 acttcctctc gccctggcaa gaacagcctc aagcttccga aaggtatcctcaagaaaaag 420 g 421 10 439 DNA RAT 10 cccctgctcc caaggaaggg cattcttaagaagtctcggc agcgtgaatc tggttactac 60 tcctctccag agcccagtga gtctggggaactcttagacg caggtgatgt gtttgtgagt 120 ggggaccccg tggagcagaa gtctccacaagcttcagggc gcctccatcg caagggcatc 180 ctcaaactca atggcaagtt ttcccgcacagccttagaag gcactgcccc tagcaccttt 240 ggctccctag accaactggc ctcccctcatcctacagccc gggccagccg tccctcggga 300 gctgtgagtg aggacagcat cctgtcctccgagtcctttg accaattgga cttgcccgag 360 cggcttcccg aaaccccact gaggagctgtgtgtctgtgg acaacctgag gaggcttgag 420 cagcctccct cagaaggcc 439 11 47 DNARAT 11 ttgggggata gccgcttttc tctgacagac tgccaagagg tgacagc 47 12 56 DNARAT 12 aaagtgaatt ttgctgcttt cggtaatgtg aatgctgtgt tctggggaaa gccact 5613 1186 DNA RAT 13 gcccggccgc tggcggaccg gctcatcaag tcgcccaaacctctgatgaa gaagcaggcg 60 gtgaagcggc accatcacaa acacaacctg aggcaccgctacgagttcct ggagaccctg 120 ggcaagggca cctacgggaa ggtgaagaaa gcacgagagagctcgggacg cctggtggcc 180 atcaagtcta tcaggaagga caaaatcaaa gatgagcaggatctgttgca cataaggagg 240 gagatcgaga tcatgtcttc actcaaccac ccccacatcattgccatcca tgaagtgttt 300 gagaacagca gcaagattgt gattgtcatg gagtacgccagccgaggcga tctgtacgat 360 tacatcagtg agcggccacg gctgaatgag cgggacgccaggcatttctt ccgacagatc 420 gtgtccgccc tgcactactg ccaccagaac gggattgttcaccgggacct caagctggag 480 aacatccttc tagatgccag tggcaacatc aagattgctgattttggcct ctccaacctg 540 tatcacaaag gcaagttcct ccagacgttc tgtgggagccctctctatgc ctcacctgag 600 atcgtcaacg ggaagcccta tgtgggccca gaggtggacagctggtctct gggcgttctt 660 ctgtacatcc tggtgcatgg caccatgccc tttgacgggcaggatcataa aaccctggtg 720 aaacaaatca gtagcggggc ttaccgagag ccgtgcaaaccgtctgatgc ctgtggcctg 780 atccggtggc tgttaatggt gaatcccatc cgtcgggccactctggagga tgtagccagt 840 cattggtggg tcaactgggg ttacagcacc cgaattggggaacaggaagc tctgcgagag 900 ggtgggcacc ctagcggtga ctctggccgg gcctctatggcggactggtt acgtcgctcc 960 tcccgccccc tcctggagaa tggagccaaa gtgtgtagcttcttcaagca gcatgtgccg 1020 ggaggtggaa gcacgggacc ggggctggag cggcaacattctcttaagaa gtcccgcaag 1080 gagaatgaca tggctcagac tctgcagaat gacccagttgaagatacttc ctctcgccct 1140 ggcaagaaca gcctcaagct tccgaaaggt atcctcaagaaaaagg 1186 14 436 DNA RAT 14 ctgctcccaa ggaagggcat tcttaagaagtctcggcagc gtgaatctgg ttactactcc 60 tctccagagc ccagtgagtc tggggaactcttagacgcag gtgatgtgtt tgtgagtggg 120 gaccccgtgg agcagaagtc tccacaagcttcagggcgcc tccatcgcaa gggcatcctc 180 aaactcaatg gcaagttttc ccgcacagccttagaaggca ctgcccctag cacctttggc 240 tccctagacc aactggcctc ccctcatcctacagcccggg ccagccgtcc ctcgggagct 300 gtgagtgagg acagcatcct gtcctccgagtcctttgacc aattggactt gcccgagcgg 360 cttcccgaaa ccccactgag gagctgtgtgtctgtggaca acctgaggag gcttgagcag 420 cctccctcag aaggcc 436 15 300 DNARAT 15 atgaagaagc aggcggtgaa gcggcaccat cacaaacaca acctgaggca ccgctacgag60 ttcctggaga ccctgggcaa gggcacctac gggaaggtga agaaagcacg agagagctcg 120ggacgcctgg tggccatcaa gtctatcagg aaggacaaaa tcaaagatga gcaggatctg 180ttgcacataa ggagggagat cgagatcatg tcttcactca accaccccca catcattgcc 240atccatgaag tgtttgagaa cagcagcaag attgtgattg tcatggagta cgccagccga 300 16169 DNA RAT 16 ggcgatctgt acgattacat cagtgagcgg ccacggctga atgagcgggacgccaggcat 60 ttcttccgac agatcgtgtc cgccctgcac tactgccacc agaacgggattgttcaccgg 120 gacctcaagc tggagaacat ccttctagat gccagtggca acatcaaga 16917 343 DNA RAT 17 gcccaaacct ctgatgaaga agcaggcggt gaagcggcac catcacaaacacaacctgag 60 gcaccgctac gagttcctgg agaccctggg caagggcacc tacgggaaggtgaagaaagc 120 acgagagagc tcgggacgcc tggtggccat caagtctatc aggaaggacaaaatcaaaga 180 tgagcaggat ctgttgcaca taaggaggga gatcgagatc atgtcttcactcaaccaccc 240 ccacatcatt gccatccatg aagtgtttga gaacagcagc aagattgtgattgtcatgga 300 gtacgccagc cgaggcgatc tgtacgatta catcagtgag cgg 343 18361 DNA RAT 18 gttcttctgt acatcctggt gcatggcacc atgccctttg acgggcaggatcataaaacc 60 ctggtgaaac aaatcagtag cggggcttac cgagagccgt gcaaaccgtctgatgcctgt 120 ggcctgatcc ggtggctgtt aatggtgaat cccatccgtc gggccactctggaggatgta 180 gccagtcatt ggtgggtcaa ctggggttac agcacccgaa ttggggaacaggaagctctg 240 cgagagggtg ggcaccctag cggtgactct ggccgggcct ctatggcggactggttacgt 300 cgctcctccc gccccctcct ggagaatgga gccaaagtgt gtagcttcttcaagcagcat 360 g 361 19 280 DNA RAT 19 gcatttcttc cgacagatcg tgtccgccctgcactactgc caccagaacg ggattgttca 60 ccgggacctc aagctggaga acatccttctagatgccagt ggcaacatca agattgctga 120 ttttggcctc tccaacctgt atcacaaaggcaagttcctc cagacgttct gtgggagccc 180 tctctatgcc tcacctgaga tcgtcaacgggaagccctat gtgggcccag aggtggacag 240 ctggtctctg ggcgttcttc tgtacatcctggtgcatggc 280 20 77 DNA RAT 20 cacccccaca tcattgccat ccatgaagtgtttgagaaca gcagcaagat tgtgattgtc 60 atggagtacg ccagccg 77 21 35 DNA RAT21 ctgatgcctg tggcctgatc cggtggctgt taatg 35 22 270 DNA RAT 22tggagaccct gggcaagggc acctacggga aggtgaagaa agcacgagag agctcgggac 60gcctggtggc catcaagtct atcaggaagg acaaaatcaa agatgagcag gatctgttgc 120acataaggag ggagatcgag atcatgtctt cactcaacca cccccacatc attgccatcc 180atgaagtgtt tgagaacagc agcaagattg tgattgtcat ggagtacgcc agccgaggcg 240atctgtacga ttacatcagt gagcggccac 270 23 226 DNA RAT 23 tatcacaaaggcaagttcct ccagacgttc tgtgggagcc ctctctatgc ctcacctgag 60 atcgtcaacgggaagcccta tgtgggccca gaggtggaca gctggtctct gggcgttctt 120 ctgtacatcctggtgcatgg caccatgccc tttgacgggc aggatcataa aaccctggtg 180 aaacaaatcagtagcggggc ttaccgagag ccgtgcaaac cgtctg 226 24 152 DNA RAT 24 tgtttgagaacagcagcaag attgtgattg tcatggagta cgccagccga ggcgatctgt 60 acgattacatcagtgagcgg ccacggctga atgagcggga cgccaggcat ttcttccgac 120 agatcgtgtccgccctgcac tactgccacc ag 152 25 66 DNA RAT 25 aacgggattg ttcaccgggacctcaagctg gagaacatcc ttctagatgc cagtggcaac 60 atcaag 66 26 123 DNA RAT26 attgctgatt ttggcctctc caacctgtat cacaaaggca agttcctcca gacgttctgt 60gggagccctc tctatgcctc acctgagatc gtcaacggga agccctatgt gggcccagag 120gtg 123 27 2026 DNA RAT 27 gtgacctctg agcctgcggc tctccgcgcg ctgctgctgctgcccgaccc cctccgcctc 60 gccgtccccg caccatggag tcggtggcct tacaccggcgcgggaacctg gctccctcgg 120 cctccgccct ggccacggag agcgcccggc cgctggcggaccggctcatc aagtcgccca 180 aacctctgat gaagaagcag gcggtgaagc ggcaccatcacaaacacaac ctgaggcacc 240 gctacgagtt cctggagacc ctgggcaagg gcacctacgggaaggtgaag aaagcacgag 300 agagctcggg acgcctggtg gccatcaagt ctatcaggaaggacaaaatc aaagatgagc 360 aggatctgtt gcacataagg agggagatcg agatcatgtcttcactcaac cacccccaca 420 tcattgccat ccatgaagtg tttgagaaca gcagcaagattgtgattgtc atggagtacg 480 ccagccgagg cgatctgtac gattacatca gtgagcggccacggctgaat gagcgggacg 540 ccaggcattt cttccgacag atcgtgtccg ccctgcactactgccaccag aacgggattg 600 ttcaccggga cctcaagctg gagaacatcc ttctagatgccagtggcaac atcaagattg 660 ctgattttgg cctctccaac ctgtatcaca aaggcaagttcctccagacg ttctgtggga 720 gccctctcta tgcctcacct gagatcgtca acgggaagccctatgtgggc ccagaggtgg 780 acagctggtc tctgggcgtt cttctgtaca tcctggtgcatggcaccatg ccctttgacg 840 ggcaggatca taaaaccctg gtgaaacaaa tcagtagcggggcttaccga gagccgtgca 900 aaccgtctga tgcctgtggc ctgatccggt ggctgttaatggtgaatccc atccgtcggg 960 ccactctgga ggatgtagcc agtcattggt gggtcaactggggttacagc acccgaattg 1020 gggaacagga agctctgcga gagggtgggc accctagcggtgactctggc cgggcctcta 1080 tggcggactg gttacgtcgc tcctcccgcc ccctcctggagaatggagcc aaagtgtgta 1140 gcttcttcaa gcagcatgtg ccgggaggtg gaagcacgggaccggggctg gagcggcaac 1200 attctcttaa gaagtcccgc aaggagaatg acatggctcagactctgcag aatgacccag 1260 ttgaagatac ttcctctcgc cctggcaaga acagcctcaagcttccgaaa ggtatcctca 1320 agaaaaaggc ctctccctca tcgggggagg tacaggagggccctcaggaa ctcagaccag 1380 tgtccaatac cccagggcag cctgtccctg ctatacccctgctcccaagg aagggcattc 1440 ttaagaagtc tcggcagcgt gaatctggtt actactcctctccagagccc agtgagtctg 1500 gggaactctt agacgcaggt gatgtgtttg tgagtggggaccccgtggag cagaagtctc 1560 cacaagcttc agggcgcctc catcgcaagg gcatcctcaaactcaatggc aagttttccc 1620 gcacagcctt agaaggcact gcccctagca cctttggctccctagaccaa ctggcctccc 1680 ctcatcctac agcccgggcc agccgtccct cgggagctgtgagtgaggac agcatcctgt 1740 cctccgagtc ctttgaccaa ttggacttgc ccgagcggcttcccgaaacc ccactgagga 1800 gctgtgtgtc tgtggacaac ctgaggaggc ttgagcagcctccctcagaa ggcctaaaac 1860 gatggtggca ggaatccttg ggggatagcc gcttttctctgacagactgc caagaggtga 1920 cagcagccta cagacaagcc ctaggaatct gctcgaagctcagctgagga ggagaggcag 1980 tgccccagtg atggggtaga ctcttagagg ggtttgcagaggaacc 2026 28 177 DNA RAT 28 cacataagtt cctgttcgca tcgaccaccagggttagaac cctgacatcc ctggaagtaa 60 tgtggagcaa cctcgcttat ttaaagaggaaacagcctct ggtttccatc tctgctgctg 120 tgcatctcaa agacctagaa agactcaactgctgtttcac ttcatctcaa ggggacc 177 29 197 DNA RAT 29 aagtgaattttgctgctttc ggtaatgtga atgctgtgtt ctggggaaag ccactgtgtc 60 attgaagtgtgtgtacagag aagtatttgg cagtgattcc ttctaatggg gggtggcctt 120 ttcagatgtatgtcttgagc actgtctgga ttgggtctcc tgtcccctca caccagaggc 180 tgtccaccctccctcat 197 30 94 DNA RAT 30 ctcagagacc tgagccttga agctgttcct gataaccagactatgatgga tatgtctgtt 60 tctcaggcca gcaggaccca gaatgtgctg actt 94 31 712DNA RAT 31 agcaagattg tgattgtcat ggagtacgcc agccgaggcg atctgtacgattacatcagt 60 gagcggccac ggctgaatga gcgggacgcc aggcatttct tccgacagatcgtgtccgcc 120 ctgcactact gccaccagaa cgggattgtt caccgggacc tcaagctggagaacatcctt 180 ctagatgcca gtggcaacat caagattgct gattttggcc tctccaacctgtatcacaaa 240 ggcaagttcc tccagacgtt ctgtgggagc cctctctatg cctcacctgagatcgtcaac 300 gggaagccct atgtgggccc agaggtggac agctggtctc tgggcgttcttctgtacatc 360 ctggtgcatg gcaccatgcc ctttgacggg caggatcata aaaccctggtgaaacaaatc 420 agtagcgggg cttaccgaga gccgtgcaaa ccgtctgatg cctgtggcctgatccggtgg 480 ctgttaatgg tgaatcccat ccgtcgggcc actctggagg atgtagccagtcattggtgg 540 gtcaactggg gttacagcac ccgaattggg gaacaggaag ctctgcgagagggtgggcac 600 cctagcggtg actctggccg ggcctctatg gcggactggt tacgtcgctcctcccgcccc 660 ctcctggaga atggagccaa agtgtgtagc ttcttcaagc agcatgtgcc gg712 32 741 DNA RAT 32 tgacctctga gcctgcggct ctccgcgcgc tgctgctgctgcccgacccc ctccgcctcg 60 ccgtccccgc accatggagt cggtggcctt acaccggcgcgggaacctgg ctccctcggc 120 ctccgccctg gccacggaga gcgcccggcc gctggcggaccggctcatca agtcgcccaa 180 acctctgatg aagaagcagg cggtgaagcg gcaccatcacaaacacaacc tgaggcaccg 240 ctacgagttc ctggagaccc tgggcaaggg cacctacgggaaggtgaaga aagcacgaga 300 gagctcggga cgcctggtgg ccatcaagtc tatcaggaaggacaaaatca aagatgagca 360 ggatctgttg cacataagga gggagatcga gatcatgtcttcactcaacc acccccacat 420 cattgccatc catgaagtgt ttgagaacag cagcaagattgtgattgtca tggagtacgc 480 cagccgaggc gatctgtacg attacatcag tgagcggccacggctgaatg agcgggacgc 540 caggcatttc ttccgacaga tcgtgtccgc cctgcactactgccaccaga acgggattgt 600 tcaccgggac ctcaagctgg agaacatcct tctagatgccagtggcaaca tcaagattgc 660 tgattttggc ctctccaacc tgtatcacaa aggcaagttcctccagacgt tctgtgggag 720 ccctctctat gcctcacctg a 741 33 660 DNA RAT 33ggtgaatccc atccgtcggg ccactctgga ggatgtagcc agtcattggt gggtcaactg 60gggttacagc acccgaattg gggaacagga agctctgcga gagggtgggc accctagcgg 120tgactctggc cgggcctcta tggcggactg gttacgtcgc tcctcccgcc ccctcctgga 180gaatggagcc aaagtgtgta gcttcttcaa gcagcatgtg ccgggaggtg gaagcacggg 240accggggctg gagcggcaac attctcttaa gaagtcccgc aaggagaatg acatggctca 300gactctgcag aatgacccag ttgaagatac ttcctctcgc cctggcaaga acagcctcaa 360gcttccgaaa ggtatcctca agaaaaaggc ctctccctca tcgggggagg tacaggaggg 420ccctcaggaa ctcagaccag tgtccaatac cccagggcag cctgtccctg ctatacccct 480gctcccaagg aagggcattc ttaagaagtc tcggcagcgt gaatctggtt actactcctc 540tccagagccc agtgagtctg gggaactctt agacgcaggt gatgtgtttg tgagtgggga 600ccccgtggag cagaagtctc cacaagcttc agggcgcctc catcgcaagg gcatcctcaa 660 34521 DNA RAT 34 tctgagcctg cggctctccg cgcgctgctg ctgctgcccg accccctccgcctcgccgtc 60 cccgcaccat ggagtcggtg gccttacacc ggcgcgggaa cctggctccctcggcctccg 120 ccctggccac ggagagcgcc cggccgctgg cggaccggct catcaagtcgcccaaacctc 180 tgatgaagaa gcaggcggtg aagcggcacc atcacaaaca caacctgaggcaccgctacg 240 agttcctgga gaccctgggc aagggcacct acgggaaggt gaagaaagcacgagagagct 300 cgggacgcct ggtggccatc aagtctatca ggaaggacaa aatcaaagatgagcaggatc 360 tgttgcacat aaggagggag atcgagatca tgtcttcact caaccacccccacatcattg 420 ccatccatga agtgtttgag aacagcagca agattgtgat tgtcatggagtacgccagcc 480 gaggcgatct gtacgattac atcagtgagc ggccacggct g 521 35 579DNA RAT 35 ataccccagg gcagcctgtc cctgctatac ccctgctccc aaggaagggcattcttaaga 60 agtctcggca gcgtgaatct ggttactact cctctccaga gcccagtgagtctggggaac 120 tcttagacgc aggtgatgtg tttgtgagtg gggaccccgt ggagcagaagtctccacaag 180 cttcagggcg cctccatcgc aagggcatcc tcaaactcaa tggcaagttttcccgcacag 240 ccttagaagg cactgcccct agcacctttg gctccctaga ccaactggcctcccctcatc 300 ctacagcccg ggccagccgt ccctcgggag ctgtgagtga ggacagcatcctgtcctccg 360 agtcctttga ccaattggac ttgcccgagc ggcttcccga aaccccactgaggagctgtg 420 tgtctgtgga caacctgagg aggcttgagc agcctccctc agaaggcctaaaacgatggt 480 ggcaggaatc cttgggggat agccgctttt ctctgacaga ctgccaagaggtgacagcag 540 cctacagaca agccctagga atctgctcga agctcagct 579 36 548 DNARAT 36 cctgctatac ccctgctccc aaggaagggc attcttaaga agtctcggca gcgtgaatct60 ggttactact cctctccaga gcccagtgag tctggggaac tcttagacgc aggtgatgtg 120tttgtgagtg gggaccccgt ggagcagaag tctccacaag cttcagggcg cctccatcgc 180aagggcatcc tcaaactcaa tggcaagttt tcccgcacag ccttagaagg cactgcccct 240agcacctttg gctccctaga ccaactggcc tcccctcatc ctacagcccg ggccagccgt 300ccctcgggag ctgtgagtga ggacagcatc ctgtcctccg agtcctttga ccaattggac 360ttgcccgagc ggcttcccga aaccccactg aggagctgtg tgtctgtgga caacctgagg 420aggcttgagc agcctccctc agaaggccta aaacgatggt ggcaggaatc cttgggggat 480agccgctttt ctctgacaga ctgccaagag gtgacagcag cctacagaca agccctagga 540atctgctc 548 37 585 DNA RAT 37 gaacaggaag ctctgcgaga gggtgggcaccctagcggtg actctggccg ggcctctatg 60 gcggactggt tacgtcgctc ctcccgccccctcctggaga atggagccaa agtgtgtagc 120 ttcttcaagc agcatgtgcc gggaggtggaagcacgggac cggggctgga gcggcaacat 180 tctcttaaga agtcccgcaa ggagaatgacatggctcaga ctctgcagaa tgacccagtt 240 gaagatactt cctctcgccc tggcaagaacagcctcaagc ttccgaaagg tatcctcaag 300 aaaaaggcct ctccctcatc gggggaggtacaggagggcc ctcaggaact cagaccagtg 360 tccaataccc cagggcagcc tgtccctgctatacccctgc tcccaaggaa gggcattctt 420 aagaagtctc ggcagcgtga atctggttactactcctctc cagagcccag tgagtctggg 480 gaactcttag acgcaggtga tgtgtttgtgagtggggacc ccgtggagca gaagtctcca 540 caagcttcag ggcgcctcca tcgcaagggcatcctcaaac tcaat 585 38 331 DNA RAT 38 acatcctggt gcatggcacc atgccctttgacgggcagga tcataaaacc ctggtgaaac 60 aaatcagtag cggggcttac cgagagccgtgcaaaccgtc tgatgcctgt ggcctgatcc 120 ggtggctgtt aatggtgaat cccatccgtcgggccactct ggaggatgta gccagtcatt 180 ggtgggtcaa ctggggttac agcacccgaattggggaaca ggaagctctg cgagagggtg 240 ggcaccctag cggtgactct ggccgggcctctatggcgga ctggttacgt cgctcctccc 300 gccccctcct ggagaatgga gccaaagtgt g331 39 164 DNA RAT 39 tggagaccct gggcaagggc acctacggga aggtgaagaaagcacgagag agctcgggac 60 gcctggtggc catcaagtct atcaggaagg acaaaatcaaagatgagcag gatctgttgc 120 acataaggag ggagatcgag atcatgtctt cactcaaccacccc 164 40 261 DNA RAT 40 ggagccctct ctatgcctca cctgagatcg tcaacgggaagccctatgtg ggcccagagg 60 tggacagctg gtctctgggc gttcttctgt acatcctggtgcatggcacc atgccctttg 120 acgggcagga tcataaaacc ctggtgaaac aaatcagtagcggggcttac cgagagccgt 180 gcaaaccgtc tgatgcctgt ggcctgatcc ggtggctgttaatggtgaat cccatccgtc 240 gggccactct ggaggatgta g 261 41 630 PRT RAT 41Met Glu Ser Val Ala Leu His Arg Arg Gly Asn Leu Ala Pro Ser Ala 1 5 1015 Ser Ala Leu Ala Thr Glu Ser Ala Arg Pro Leu Ala Asp Arg Leu Ile 20 2530 Lys Ser Pro Lys Pro Leu Met Lys Lys Gln Ala Val Lys Arg His His 35 4045 His Lys His Asn Leu Arg His Arg Tyr Glu Phe Leu Glu Thr Leu Gly 50 5560 Lys Gly Thr Tyr Gly Lys Val Lys Lys Ala Arg Glu Ser Ser Gly Arg 65 7075 80 Leu Val Ala Ile Lys Ser Ile Arg Lys Asp Lys Ile Lys Asp Glu Gln 8590 95 Asp Leu Leu His Ile Arg Arg Glu Ile Glu Ile Met Ser Ser Leu Asn100 105 110 His Pro His Ile Ile Ala Ile His Glu Val Phe Glu Asn Ser SerLys 115 120 125 Ile Val Ile Val Met Glu Tyr Ala Ser Arg Gly Asp Leu TyrAsp Tyr 130 135 140 Ile Ser Glu Arg Pro Arg Leu Asn Glu Arg Asp Ala ArgHis Phe Phe 145 150 155 160 Arg Gln Ile Val Ser Ala Leu His Tyr Cys HisGln Asn Gly Ile Val 165 170 175 His Arg Asp Leu Lys Leu Glu Asn Ile LeuLeu Asp Ala Ser Gly Asn 180 185 190 Ile Lys Ile Ala Asp Phe Gly Leu SerAsn Leu Tyr His Lys Gly Lys 195 200 205 Phe Leu Gln Thr Phe Cys Gly SerPro Leu Tyr Ala Ser Pro Glu Ile 210 215 220 Val Asn Gly Lys Pro Tyr ValGly Pro Glu Val Asp Ser Trp Ser Leu 225 230 235 240 Gly Val Leu Leu TyrIle Leu Val His Gly Thr Met Pro Phe Asp Gly 245 250 255 Gln Asp His LysThr Leu Val Lys Gln Ile Ser Ser Gly Ala Tyr Arg 260 265 270 Glu Pro CysLys Pro Ser Asp Ala Cys Gly Leu Ile Arg Trp Leu Leu 275 280 285 Met ValAsn Pro Ile Arg Arg Ala Thr Leu Glu Asp Val Ala Ser His 290 295 300 TrpTrp Val Asn Trp Gly Tyr Ser Thr Arg Ile Gly Glu Gln Glu Ala 305 310 315320 Leu Arg Glu Gly Gly His Pro Ser Gly Asp Ser Gly Arg Ala Ser Met 325330 335 Ala Asp Trp Leu Arg Arg Ser Ser Arg Pro Leu Leu Glu Asn Gly Ala340 345 350 Lys Val Cys Ser Phe Phe Lys Gln His Val Pro Gly Gly Gly SerThr 355 360 365 Gly Pro Gly Leu Glu Arg Gln His Ser Leu Lys Lys Ser ArgLys Glu 370 375 380 Asn Asp Met Ala Gln Thr Leu Gln Asn Asp Pro Val GluAsp Thr Ser 385 390 395 400 Ser Arg Pro Gly Lys Asn Ser Leu Lys Leu ProLys Gly Ile Leu Lys 405 410 415 Lys Lys Ala Ser Pro Ser Ser Gly Glu ValGln Glu Gly Pro Gln Glu 420 425 430 Leu Arg Pro Val Ser Asn Thr Pro GlyGln Pro Val Pro Ala Ile Pro 435 440 445 Leu Leu Pro Arg Lys Gly Ile LeuLys Lys Ser Arg Gln Arg Glu Ser 450 455 460 Gly Tyr Tyr Ser Ser Pro GluPro Ser Glu Ser Gly Glu Leu Leu Asp 465 470 475 480 Ala Gly Asp Val PheVal Ser Gly Asp Pro Val Glu Gln Lys Ser Pro 485 490 495 Gln Ala Ser GlyArg Leu His Arg Lys Gly Ile Leu Lys Leu Asn Gly 500 505 510 Lys Phe SerArg Thr Ala Leu Glu Gly Thr Ala Pro Ser Thr Phe Gly 515 520 525 Ser LeuAsp Gln Leu Ala Ser Pro His Pro Thr Ala Arg Ala Ser Arg 530 535 540 ProSer Gly Ala Val Ser Glu Asp Ser Ile Leu Ser Ser Glu Ser Phe 545 550 555560 Asp Gln Leu Asp Leu Pro Glu Arg Leu Pro Glu Thr Pro Leu Arg Ser 565570 575 Cys Val Ser Val Asp Asn Leu Arg Arg Leu Glu Gln Pro Pro Ser Glu580 585 590 Gly Leu Lys Arg Trp Trp Gln Glu Ser Leu Gly Asp Ser Arg PheSer 595 600 605 Leu Thr Asp Cys Gln Glu Val Thr Ala Ala Tyr Arg Gln AlaLeu Gly 610 615 620 Ile Cys Ser Lys Leu Ser 625 630 42 59 PRT RAT 42 AlaArg Pro Leu Ala Asp Arg Leu Ile Lys Ser Pro Lys Pro Leu Met 1 5 10 15Lys Lys Gln Ala Val Lys Arg His His His Lys His Asn Leu Arg His 20 25 30Arg Tyr Glu Phe Leu Glu Thr Leu Gly Lys Gly Thr Tyr Gly Lys Val 35 40 45Lys Lys Ala Arg Glu Ser Ser Gly Arg Leu Val 50 55 43 40 PRT RAT 43 ValAla Ile Lys Ser Ile Arg Lys Asp Lys Ile Lys Asp Glu Gln Asp 1 5 10 15Leu Leu His Ile Arg Arg Glu Ile Glu Ile Met Ser Ser Leu Asn His 20 25 30Pro His Ile Ile Ala Ile His Glu 35 40 44 49 PRT RAT 44 Val Phe Glu AsnSer Ser Lys Ile Val Ile Val Met Glu Tyr Ala Ser 1 5 10 15 Arg Gly AspLeu Tyr Asp Tyr Ile Ser Glu Arg Pro Arg Leu Asn Glu 20 25 30 Arg Asp AlaArg His Phe Phe Arg Gln Ile Val Ser Ala Leu His Tyr 35 40 45 Cys 45 35PRT RAT 45 Ile Ala Asp Phe Gly Leu Ser Asn Leu Tyr His Lys Gly Lys PheLeu 1 5 10 15 Gln Thr Phe Cys Gly Ser Pro Leu Tyr Ala Ser Pro Glu IleVal Asn 20 25 30 Gly Lys Pro 35 46 39 PRT RAT 46 Val Asp Ser Trp Ser LeuGly Val Leu Leu Tyr Ile Leu Val His Gly 1 5 10 15 Thr Met Pro Phe AspGly Gln Asp His Lys Thr Leu Val Lys Gln Ile 20 25 30 Ser Ser Gly Ala TyrArg Glu 35 47 140 PRT RAT 47 Asp Ala Cys Gly Leu Ile Arg Trp Leu Leu MetVal Asn Pro Ile Arg 1 5 10 15 Arg Ala Thr Leu Glu Asp Val Ala Ser HisTrp Trp Val Asn Trp Gly 20 25 30 Tyr Ser Thr Arg Ile Gly Glu Gln Glu AlaLeu Arg Glu Gly Gly His 35 40 45 Pro Ser Gly Asp Ser Gly Arg Ala Ser MetAla Asp Trp Leu Arg Arg 50 55 60 Ser Ser Arg Pro Leu Leu Glu Asn Gly AlaLys Val Cys Ser Phe Phe 65 70 75 80 Lys Gln His Val Pro Gly Gly Gly SerThr Gly Pro Gly Leu Glu Arg 85 90 95 Gln His Ser Leu Lys Lys Ser Arg LysGlu Asn Asp Met Ala Gln Thr 100 105 110 Leu Gln Asn Asp Pro Val Glu AspThr Ser Ser Arg Pro Gly Lys Asn 115 120 125 Ser Leu Lys Leu Pro Lys GlyIle Leu Lys Lys Lys 130 135 140 48 146 PRT RAT 48 Pro Leu Leu Pro ArgLys Gly Ile Leu Lys Lys Ser Arg Gln Arg Glu 1 5 10 15 Ser Gly Tyr TyrSer Ser Pro Glu Pro Ser Glu Ser Gly Glu Leu Leu 20 25 30 Asp Ala Gly AspVal Phe Val Ser Gly Asp Pro Val Glu Gln Lys Ser 35 40 45 Pro Gln Ala SerGly Arg Leu His Arg Lys Gly Ile Leu Lys Leu Asn 50 55 60 Gly Lys Phe SerArg Thr Ala Leu Glu Gly Thr Ala Pro Ser Thr Phe 65 70 75 80 Gly Ser LeuAsp Gln Leu Ala Ser Pro His Pro Thr Ala Arg Ala Ser 85 90 95 Arg Pro SerGly Ala Val Ser Glu Asp Ser Ile Leu Ser Ser Glu Ser 100 105 110 Phe AspGln Leu Asp Leu Pro Glu Arg Leu Pro Glu Thr Pro Leu Arg 115 120 125 SerCys Val Ser Val Asp Asn Leu Arg Arg Leu Glu Gln Pro Pro Ser 130 135 140Glu Gly 145 49 15 PRT RAT 49 Leu Gly Asp Ser Arg Phe Ser Leu Thr Asp CysGln Glu Val Thr 1 5 10 15 50 251 PRT RAT 50 Tyr Glu Phe Leu Glu Thr LeuGly Lys Gly Thr Tyr Gly Lys Val Lys 1 5 10 15 Lys Ala Arg Glu Ser SerGly Arg Leu Val Ala Ile Lys Ser Ile Arg 20 25 30 Lys Asp Lys Ile Lys AspGlu Gln Asp Leu Leu His Ile Arg Arg Glu 35 40 45 Ile Glu Ile Met Ser SerLeu Asn His Pro His Ile Ile Ala Ile His 50 55 60 Glu Val Phe Glu Asn SerSer Lys Ile Val Ile Val Met Glu Tyr Ala 65 70 75 80 Ser Arg Gly Asp LeuTyr Asp Tyr Ile Ser Glu Arg Pro Arg Leu Asn 85 90 95 Glu Arg Asp Ala ArgHis Phe Phe Arg Gln Ile Val Ser Ala Leu His 100 105 110 Tyr Cys His GlnAsn Gly Ile Val His Arg Asp Leu Lys Leu Glu Asn 115 120 125 Ile Leu LeuAsp Ala Ser Gly Asn Ile Lys Ile Ala Asp Phe Gly Leu 130 135 140 Ser AsnLeu Tyr His Lys Gly Lys Phe Leu Gln Thr Phe Cys Gly Ser 145 150 155 160Pro Leu Tyr Ala Ser Pro Glu Ile Val Asn Gly Lys Pro Tyr Val Gly 165 170175 Pro Glu Val Asp Ser Trp Ser Leu Gly Val Leu Leu Tyr Ile Leu Val 180185 190 His Gly Thr Met Pro Phe Asp Gly Gln Asp His Lys Thr Leu Val Lys195 200 205 Gln Ile Ser Ser Gly Ala Tyr Arg Glu Pro Cys Lys Pro Ser AspAla 210 215 220 Cys Gly Leu Ile Arg Trp Leu Leu Met Val Asn Pro Ile ArgArg Ala 225 230 235 240 Thr Leu Glu Asp Val Ala Ser His Trp Trp Val 245250 51 177 DNA Homo sapiens 51 gcccggccgc tggcggaagg gctgatcaagtcgcccaagc ccctaatgaa gaagcaggcg 60 gtgaagcggc accaccacaa gcacaacctgcggcaccgct acgagttcct ggagaccctg 120 ggcaaaggca cctacgggaa ggtgaagaaggcgcgggaga gctcggggcg cctggtg 177 52 122 DNA Homo sapiens 52 ggtggccatcaagtcaatcc ggaaggacaa aatcaaagat gagcaagatc tgatgcacat 60 acggagggagattgagatca tgtcatcact caaccaccct cacatcattg ccatccatga 120 ag 122 53 150DNA Homo sapiens 53 agtgtttgag aacagcagca agatcgtgat cgtcatggagtatgccagcc ggggcgacct 60 ttatgactac atcagcgagc ggcagcagct cagtgagcgcgaagctaggc atttcttccg 120 gcagatcgtc tctgccgtgc actattgcca 150 54 109DNA Homo sapiens 54 agattgctga cttcggtctc tccaacctct accatcaaggcaagttcctg cagacattct 60 gtgggagccc cctctatgcc tcgccagaga ttgtcaatgggaagcccta 109 55 121 DNA Homo sapiens 55 aggtggacag ctggtccctgggtgttctcc tctacatcct ggtgcatggc accatgccct 60 ttgatgggca tgaccataagatcctagtga aacagatcag caacggggcc taccgggagc 120 c 121 56 421 DNA Homosapiens 56 gatgcctgtg gcctgatccg gtggctgttg atggtgaacc ccacccgccgggccaccctg 60 gaggatgtgg ccagtcactg gtgggtcaac tggggctacg ccacccgagtgggagagcag 120 gaggctccgc atgagggtgg gcaccctggc agtgactctg cccgcgcctccatggctgac 180 tggctccggc gttcctcccg ccccctcctg gagaatgggg ccaaggtgtgcagcttcttc 240 aagcagcatg cacctggtgg gggaagcacc acccctggcc tggagcgccagcattcgctc 300 aagaagtccc gcaaggagaa tgacatggcc cagtctctcc acagtgacacggctgatgac 360 actgcccatc gccctggcaa gagcaacctc aagctgccaa agggcattctcaagaagaag 420 g 421 57 442 DNA Homo sapiens 57 cccctgctcc ccaagaagggcattctcaag aagccccgac agcgcgagtc tggctactac 60 tcctctcccg agcccagtgaatctggggag ctcttggacg caggcgacgt gtttgtgagt 120 ggggatccca aggagcagaagcctccgcaa gcttcagggc tgctcctcca tcgcaaaggc 180 atcctcaaac tcaatggcaagttctcccag acagccttgg agctcgcggc ccccaccacc 240 ttcggctccc tggatgaactcgccccacct cgccccctgg cccgggccag ccgaccctca 300 ggggctgtga gcgaggacagcatcctgtcc tctgagtcct ttgaccagct ggacttgcct 360 gaacggctcc cagagcccccactgcggggc tgtgtgtctg tggacaacct cacggggctt 420 gaggagcccc cctcagaggg cc442 58 47 DNA Homo sapiens 58 ttgggggaca gctgcttttc cctgacagactgccaggagg tgacagc 47 59 56 DNA Homo sapiens 59 aaagtgaatc ttgctgttttcaataatgtg aatgctatgt tctgggaaaa tccact 56 60 11 DNA Homo sapiens 60cacaggccca g 11 61 13 DNA Homo sapiens 61 acctaaaccc tct 13 62 59 PRTHomo sapiens 62 Ala Arg Pro Leu Ala Glu Gly Leu Ile Lys Ser Pro Lys ProLeu Met 1 5 10 15 Lys Lys Gln Ala Val Lys Arg His His His Lys His AsnLeu Arg His 20 25 30 Arg Tyr Glu Phe Leu Glu Thr Leu Gly Lys Gly Thr TyrGly Lys Val 35 40 45 Lys Lys Ala Arg Glu Ser Ser Gly Arg Leu Val 50 5563 40 PRT Homo sapiens 63 Val Ala Ile Lys Ser Ile Arg Lys Asp Lys IleLys Asp Glu Gln Asp 1 5 10 15 Leu Met His Ile Arg Arg Glu Ile Glu IleMet Ser Ser Leu Asn His 20 25 30 Pro His Ile Ile Ala Ile His Glu 35 4064 49 PRT Homo sapiens 64 Val Phe Glu Asn Ser Ser Lys Ile Val Ile ValMet Glu Tyr Ala Ser 1 5 10 15 Arg Gly Asp Leu Tyr Asp Tyr Ile Ser GluArg Gln Gln Leu Ser Glu 20 25 30 Arg Glu Ala Arg His Phe Phe Arg Gln IleVal Ser Ala Val His Tyr 35 40 45 Cys 65 35 PRT Homo sapiens 65 Ile AlaAsp Phe Gly Leu Ser Asn Leu Tyr His Gln Gly Lys Phe Leu 1 5 10 15 GlnThr Phe Cys Gly Ser Pro Leu Tyr Ala Ser Pro Glu Ile Val Asn 20 25 30 GlyLys Pro 35 66 39 PRT Homo sapiens 66 Val Asp Ser Trp Ser Leu Gly Val LeuLeu Tyr Ile Leu Val His Gly 1 5 10 15 Thr Met Pro Phe Asp Gly His AspHis Lys Ile Leu Val Lys Gln Ile 20 25 30 Ser Asn Gly Ala Tyr Arg Glu 3567 140 PRT Homo sapiens 67 Asp Ala Cys Gly Leu Ile Arg Trp Leu Leu MetVal Asn Pro Thr Arg 1 5 10 15 Arg Ala Thr Leu Glu Asp Val Ala Ser HisTrp Trp Val Asn Trp Gly 20 25 30 Tyr Ala Thr Arg Val Gly Glu Gln Glu AlaPro His Glu Gly Gly His 35 40 45 Pro Gly Ser Asp Ser Ala Arg Ala Ser MetAla Asp Trp Leu Arg Arg 50 55 60 Ser Ser Arg Pro Leu Leu Glu Asn Gly AlaLys Val Cys Ser Phe Phe 65 70 75 80 Lys Gln His Ala Pro Gly Gly Gly SerThr Thr Pro Gly Leu Glu Arg 85 90 95 Gln His Ser Leu Lys Lys Ser Arg LysGlu Asn Asp Met Ala Gln Ser 100 105 110 Leu His Ser Asp Thr Ala Asp AspThr Ala His Arg Pro Gly Lys Ser 115 120 125 Asn Leu Lys Leu Pro Lys GlyIle Leu Lys Lys Lys 130 135 140 68 147 PRT Homo sapiens 68 Pro Leu LeuPro Lys Lys Gly Ile Leu Lys Lys Pro Arg Gln Arg Glu 1 5 10 15 Ser GlyTyr Tyr Ser Ser Pro Glu Pro Ser Glu Ser Gly Glu Leu Leu 20 25 30 Asp AlaGly Asp Val Phe Val Ser Gly Asp Pro Lys Glu Gln Lys Pro 35 40 45 Pro GlnAla Ser Gly Leu Leu Leu His Arg Lys Gly Ile Leu Lys Leu 50 55 60 Asn GlyLys Phe Ser Gln Thr Ala Leu Glu Leu Ala Ala Pro Thr Thr 65 70 75 80 PheGly Ser Leu Asp Glu Leu Ala Pro Pro Arg Pro Leu Ala Arg Ala 85 90 95 SerArg Pro Ser Gly Ala Val Ser Glu Asp Ser Ile Leu Ser Ser Glu 100 105 110Ser Phe Asp Gln Leu Asp Leu Pro Glu Arg Leu Pro Glu Pro Pro Leu 115 120125 Arg Gly Cys Val Ser Val Asp Asn Leu Thr Gly Leu Glu Glu Pro Pro 130135 140 Ser Glu Gly 145 69 15 PRT Homo sapiens 69 Leu Gly Asp Ser CysPhe Ser Leu Thr Asp Cys Gln Glu Val Thr 1 5 10 15 70 1186 DNA Homosapiens 70 gcccggccgc tggcggaagg gctgatcaag tcgcccaagc ccctaatgaagaagcaggcg 60 gtgaagcggc accaccacaa gcacaacctg cggcaccgct acgagttcctggagaccctg 120 ggcaaaggca cctacgggaa ggtgaagaag gcgcgggaga gctcggggcgcctggtggcc 180 atcaagtcaa tccggaagga caaaatcaaa gatgagcaag atctgatgcacatacggagg 240 gagattgaga tcatgtcatc actcaaccac cctcacatca ttgccatccatgaagtgttt 300 gagaacagca gcaagatcgt gatcgtcatg gagtatgcca gccggggcgacctttatgac 360 tacatcagcg agcggcagca gctcagtgag cgcgaagcta ggcatttcttccggcagatc 420 gtctctgccg tgcactattg ccatcagaac agagttgtcc accgagatctcaagctggag 480 aacatcctct tggatgccaa tgggaatatc aagattgctg acttcggcctctccaacctc 540 taccatcaag gcaagttcct gcagacattc tgtgggagcc ccctctatgcctcgccagag 600 attgtcaatg ggaagcccta cacaggccca gaggtggaca gctggtccctgggtgttctc 660 ctctacatcc tggtgcatgg caccatgccc tttgatgggc atgaccataagatcctagtg 720 aaacagatca gcaacggggc ctaccgggag ccacctaaac cctctgatgcctgtggcctg 780 atccggtggc tgttgatggt gaaccccacc cgccgggcca ccctggaggatgtggccagt 840 cactggtggg tcaactgggg ctacgccacc cgagtgggag agcaggaggctccgcatgag 900 ggtgggcacc ctggcagtga ctctgcccgc gcctccatgg ctgactggctccggcgttcc 960 tcccgccccc tcctggagaa tggggccaag gtgtgcagct tcttcaagcagcatgcacct 1020 ggtgggggaa gcaccacccc tggcctggag cgccagcatt cgctcaagaagtcccgcaag 1080 gagaatgaca tggcccagtc tctccacagt gacacggctg atgacactgcccatcgccct 1140 ggcaagagca acctcaagct gccaaagggc attctcaaga agaagg 118671 439 DNA Homo sapiens 71 ctgctcccca agaagggcat tctcaagaag ccccgacagcgcgagtctgg ctactactcc 60 tctcccgagc ccagtgaatc tggggagctc ttggacgcaggcgacgtgtt tgtgagtggg 120 gatcccaagg agcagaagcc tccgcaagct tcagggctgctcctccatcg caaaggcatc 180 ctcaaactca atggcaagtt ctcccagaca gccttggagctcgcggcccc caccaccttc 240 ggctccctgg atgaactcgc cccacctcgc cccctggcccgggccagccg accctcaggg 300 gctgtgagcg aggacagcat cctgtcctct gagtcctttgaccagctgga cttgcctgaa 360 cggctcccag agcccccact gcggggctgt gtgtctgtggacaacctcac ggggcttgag 420 gagcccccct cagagggcc 439 72 47 DNA Homosapiens 72 ttgggggaca gctgcttttc cctgacagac tgccaggagg tgacagc 47 73 56DNA Homo sapiens 73 aaagtgaatc ttgctgtttt caataatgtg aatgctatgttctgggaaaa tccact 56 74 300 DNA Homo sapiens 74 atgaagaagc aggcggtgaagcggcaccac cacaagcaca acctgcggca ccgctacgag 60 ttcctggaga ccctgggcaaaggcacctac gggaaggtga agaaggcgcg ggagagctcg 120 gggcgcctgg tggccatcaagtcaatccgg aaggacaaaa tcaaagatga gcaagatctg 180 atgcacatac ggagggagattgagatcatg tcatcactca accaccctca catcattgcc 240 atccatgaag tgtttgagaacagcagcaag atcgtgatcg tcatggagta tgccagccgg 300 75 169 DNA Homo sapiens75 ggcgaccttt atgactacat cagcgagcgg cagcagctca gtgagcgcga agctaggcat 60ttcttccggc agatcgtctc tgccgtgcac tattgccatc agaacagagt tgtccaccga 120gatctcaagc tggagaacat cctcttggat gccaatggga atatcaaga 169 76 341 DNAHomo sapiens 76 gcccaagccc ctaatgaaga agcaggcggt gaagcggcac caccacaagcacaacctgcg 60 gcaccgtacg agttcctgga gaccctgggc aaaggcacct acgggaaggtgaagaaggcg 120 cgggagagct cggggcgcct ggtggccatc aagtcaatcc ggaagacaaaatcaaagatg 180 agcaagatct gatgcacata cggagggaga ttgagatcat gtcatcactcaaccaccctc 240 acatcattgc catccatgaa gtgtttgaga acagcagcaa gatcgtgatcgtcatggagt 300 atgccagccg gggcgacctt tatgactaca tcagcgagcg g 341 77 356DNA Homo sapiens 77 gttctcctct acatcctggt gcatggcacc atgcccttgatgggcatgac cataagatcc 60 tagtgaaacg agatcagcaa cggggcctac cgggagcccacctaaacgct ctgatgcctg 120 tggcctgatc cggtggctgt tgatggtgaa ccccagccgccgggcaccct ggaggatgtg 180 gccagtcact ggtgggtcaa ctggggctac gccacccgagtgggagagca ggaggctccg 240 catgagggtg ggcaccctgg cagtgactct gcccgcgctccatggctgac tgctccggcg 300 tcctcccgcc cctcctgaga atggggccaa ggtgtgcagcttcttcaagc agcatg 356 78 282 DNA Homo sapiens 78 gcatttcttc cggcagatcgtctctgccgt gcacctattg ccatcagaac agagttgtcc 60 accgagatct caagctggagaacatcctct tggatgccaa tgggaatatc aagattgctg 120 acttcggcct ctccaacctctaccatcaag gcaagttcct gcagacattc tgtgggagcc 180 ccctctatgc ctcgccagagatttgtcaat gggaagccta cacaggccca gaggttggac 240 agctggtccc tgggtgttctcctctacatc ctggtgcatg gc 282 79 78 DNA Homo sapiens 79 caccctcacatcatctgcca tccatgaagt gtttgagaac agcagcaaga tcgtgatcgt 60 catggagtatgccagccg 78 80 35 DNA Homo sapiens misc_feature “n” equal anyone of thethe four possibilities a, c, t, or g 80 ctgatngctg tggcttgatc cggtggctgttnatg 35 81 270 DNA Homo sapiens 81 tggagaccct gggcaaaggc acctacgggaaggtgaagaa ggcgcgggag agctcggggc 60 gcctggtggc catcaagtca atccggaaggacaaaatcaa agatgagcaa gatctgatgc 120 acatacggag ggagattgag atcatgtcatcactcaacca ccctcacatc attgccatcc 180 atgaagtgtt tgagaacagc agcaagatcgtgatcgtcat ggagtatgcc agcgggggcg 240 acttttatga ctacgtcagc ggggcggcag270 82 226 DNA Homo sapiens 82 taccatcaag gcaagttcct gcagacattctgtgggagcc ccctctatgc ctcgccagag 60 attgtcaatg ggaagcccta cacaggcccagaggtggaca gctggtccct gggtgttctc 120 ctctacatcc ctgtgcatgg caccatgccctttgatgggc atgaccataa gatcctagtg 180 aaacagatca gcaacggggc ctaccgggagccaactaaac cctctg 226 83 122 DNA MOUSE 83 ggtggccatc aagtccatcaggaaagacaa aatcaaagat gagcaggatc tgctgcacat 60 acggagggag attgagatcatgtcttcact caaccacccc cacatcattg ccatccatga 120 ag 122 84 152 DNA MOUSE84 tgtttgagaa tagcagcaag attgtgattg tcatggagta tgccagccga ggcgatctgt 60atgattacat cagtgagcgg ccacggctga gtgagcggga cgccaggcat ttcttccgac 120agatcgtgtc tgccctgcac tactgccacc ag 152 85 66 DNA MOUSE 85 aacgggatcgttcaccgaga tctcaagctg gaaaacatcc ttctagatgc caatggaaac 60 atcaag 66 86123 DNA MOUSE 86 attgctgact ttggcctctc caacctgtac cacaaaggca agttcctccagacgttctgt 60 gggagccctc tctacgcctc gcctgagata gtcaacggga agccctatgtgggcccagag 120 gtg 123 87 2027 DNA MOUSE 87 gtgacctctg agcccgcggctcagcgcgcg ctgctactgc tgcccgaccc actccacctc 60 gcggtccccg caccatggagtcggtggcct tactccagcg cccgagccag gctccctcgg 120 cctccgccct ggcctcggagagcgcccggc cgctggcgga cgggctcatc aagtcgccta 180 aacctctgat gaagaagcaggcggtgaagc ggcaccatca caaacacaac ctgcggcacc 240 gctacgagtt cctggagacgctgggcaagg gcacctacgg gaaggtgaag aaggcacgag 300 agagctcggg gcgtctggtggccatcaagt ccatcaggaa agacaaaatc aaagatgagc 360 aggatctgtt gcacataaggagggagatcg agatcatgtc ttcactcaac cacccccaca 420 tcattgccat ccatgaagtgtttgagaata gcagcaagat tgtgattgtc atggagtatg 480 ccagccgagg cgatctgtatgattacatca gtgagcggcc acggctgagt gagcgggacg 540 ccaggcattt cttccgacagatcgtgtctg ccctgcacta ctgccaccag aacgggatcg 600 ttcaccgaga tctcaagctggaaaacatcc ttctagatgc caatggaaac atcaagattg 660 ctgactttgg cctctccaacctgtaccaca aaggcaagtt cctccagacg ttctgtggga 720 gccctctcta cgcctcgcctgagatagtca acgggaagcc ctatgtgggc ccagaggtgg 780 acagctggtc tctgggcgttctcctgtaca tcctggtgca tggcaccatg ccctttgacg 840 ggcaggatca taaaacactggtgaagcaaa tcagtaacgg ggcttaccgt gagccgccca 900 agccgtccga tgcctgtggcctgatccggt ggctgttaat ggtgaacccc acccgtcggg 960 ccacactgga ggatgtagccagtcattggt gggtcaactg gggttacacc accggagtcg 1020 gggaacagga agccctgcgtgagggtgggc accctagtgg tgactttggc cgggcctcca 1080 tggcggactg gttacgtcgctcctcgcgcc ccctcctgga gaatggagcc aaggtgtgca 1140 gcttcttcaa gcagcacgtgccgggaggtg gaagcactgt acctgggctg gagcggcaac 1200 attctcttaa gaagtcccgaaaggagaatg acatggctca aaatctgcaa ggtgacccgg 1260 ctgaggatac ctcttctcgccctggcaaga gcagccttaa gcttccgaaa ggcattctca 1320 agaaaaagtc ctctacctcgtcaggggagg tacaggagga ccctcaggaa ctcagaccgg 1380 tgcctgatac tccagggcagcctgtccctg ctgtatccct gctcccaagg aaaggcatcc 1440 ttaagaagtc tcgacagcgtgaatctggtt actactcctc tccagagccc agcgagtctg 1500 gggaactctt agacgccagtgatgtgtttg tgagtgggga ccccgtggag cagaagtctc 1560 cacaggcttc agggctcctcctccaccgca agggcattct caaactcaat ggcaagttct 1620 cccgcacagc cttagaaggcactaccccta gcacctttgg ctccctggac caactggcct 1680 cctcccatcc tgcagcccggcccagccgcc cctcaggggc tgtgagtgag gacagcatcc 1740 tgtcctccga gtcctttgaccaattggact tgcctgaacg tcttcccgaa accccactga 1800 ggggctgtgt gtctgtggacaacctgaggg ggcttgagca gcctccctca gaaggtctga 1860 agcgatggtg gcaggaatccttgggggata gctgcttttc tctgacagac tgccaagagg 1920 tgactgcagc ctacagacaagccctaggaa tctgctcaaa gctcagctga ggaagggaga 1980 tggtgcccta gtatggggtaggctctgaga gggtttgcag aggaacc 2027 88 178 DNA MOUSE 88 cacataagtttctgtttcca tcaaccacca gggttagaac cctgacttcc tgggaggtaa 60 tgtgtagtgactgccattat ttagagagga aacagcctct ggtttccatc tctgctgctg 120 tgcatctcaaagacctggga agactcggac cgctgtttga cttcatctca aggggacc 178 89 205 DNAMOUSE 89 aagtgaattt tgctgctttc aataatgtga atgctgtgtt ctggggaactccactgtgcc 60 actgaagttt atgtacagag aagtatttgg caatgatgtc cctctattcaaggggggtgg 120 gggcgttttt caaatgtatg tcttgagcac tgtctggatt gagtctccagtcccttcaca 180 cccaaggctg gccaccctcc ctcat 205 90 93 DNA MOUSE 90ctcagagact tgaaccttga agctgttcct agtacccaga tgtggatgga tgctctgttt 60ctcaggccaa cgggacctag aatgtgctga ctt 93 91 707 DNA MOUSE 91 agcaagattgtgattgtcat ggagtatgcc agccgaggcg atctgtatga ttacatcagt 60 gagcggccacggctgagtga gcgggacgcc aggcatttct tccgacagat cgtgtctgcc 120 ctgcactactgccaccagaa cgggatcgtt caccgagatc tcaagctgga aaacatcctt 180 ctagatgccaatggaaacat caagattgct gactttggcc tctccaacct gtaccacaaa 240 ggcaagttcctccagacgtt ctgtgggagc cctctctacg cctcgcctga gatagtcaac 300 gggaagccctatgtgggccc agaggtggac agctggtctc tgggcgttct cctgtacatc 360 ctggtgcatggcaccatgcc ctttgacggg caggatcata aaacactggt gaagcaaatc 420 agtaacggggcttaccgtga gccgcccaag ccgtccgatg cctgtggcct gatccggtgg 480 ctgttaatggtgaaccccac ccgtcgggcc acactggagg atgtagccag tcattggtgg 540 gtcaactggggttacaccac cggagtcggg gaacaggaag ccctgcgtga gggtgggcac 600 cctagtggtgactttggcgg gctccatggc ggactggtta cgtcgctctc gcgcccctcc 660 tggagaatgggccacagtgt gcagttcttc aagccagcac gtgccgg 707 92 734 DNA MOUSE 92tgacctctga gcccgcggct cagcgcgcgc tgctactgtg cccgaccact ccacctcgcg 60gtccccgcac catggagtcg gtggccttac tccagcgccc gagccaggct ccctcggcct 120ccgccctggc ctcggagagc gcccggccgc tggcggacgg gctcatcaag tcgcctaaac 180ctctgatgaa gaagcaggcg gtgaagcggc accatcacaa acacaacctg cggcaccgct 240acgagttcct ggagacgctg ggcaagggca cctacgggaa ggtgaagaag gcacgagaga 300gctcggggcg tctggtggcc atcaagtcca tcaggaaaga caaaatcaaa gatgagcagg 360atctgctgca catacggagg gagatgagat catgtcttca ctcaaccacc cccacatcat 420tgccatccat gaagtgtttg agaatagcag caagattgtg attgtcatgg agtatgccag 480ccgaggcgat ctgtatgatt acatcagtga gcggcacggc tgagtgagcg ggacgccagg 540catttcttcc gacagatcgt gtctgcctgc actactgcca ccagaacggg atcgttcacc 600gagatctcaa gctggaaaac atccttctag atgccaatgg aaacatcaag atgctgactt 660gggctctcca aacctgtacc acaagggcca gttgctccag acgtctgggg gagccctctc 720tacgcctcgc ctga 734 93 661 DNA MOUSE 93 ggtgaacccc acccgtcggg ccacactggaggatgtagcc agtcattggt gggtcaactg 60 gggttacacc accggagtcg gggaacaggaagccctgcgt gagggtgggc accctagtgg 120 tgactttggc cgggcctcca tggcggactggttacgtcgc tcctcgcgcc ccctcctgga 180 gaatggagcc aaggtgtgca gcttcttcaagcagcacgtg ccgggaggtg gaagcactgt 240 acctgggctg gagcggcaac attctcttaagaagtcccga aaggagaatg acatggctca 300 aaatctgcaa ggtgacccgg ctgaggatacctcttctcgc cctggcaaga gcagccttaa 360 gcttccgaaa ggcattctca agaaaaagtcctctacctcg tcaggggagg tacaggagga 420 ccctcaggaa ctcagaccgg tgcctgatactccagggcag cctgtccctg ctgtatccct 480 gctcccaagg aaaggcatcc ttaagaagtctcgacagcgt gaatctggtt actactcctc 540 tccagagccc agcgagtctg gggaactcttagacgccagt gatgtgtttg tgagtggggg 600 ccccgtggag cagaagtctc cacaggcttcaggctctcct ccaccgcaag ggcattctca 660 a 661 94 521 DNA MOUSE 94tctgagcccg cggctctccg cgcgctgcta ctgctgcccg acccactcca cctcgcggtc 60cccgcaccat ggagtcggtg gccttactcc agcgcccgag ccaggctccc tcggcctccg 120ccctggcctc ggagagcgcc cggccgctgg cggacgggct catcaagtcg cctaaacctc 180tgatgaagaa gcaggcggtg aagcggcacc atcacaaaca caacctgcgg caccgctacg 240agttcctgga gaccctgggc aagggcacct acgggaaggt gaagaaggca cgagagagct 300cggggcgtct ggtggccatc aagtcaatca ggaaagacaa aatcaaagat gagcaggatc 360tgctgcacat acggagggag attgagatca tgtcttcact caaccacccc cacatcattg 420ccatccatga agtgtttgag aatagcagca agattgtgat tgtcatggag tatgccagcc 480gaggcgatct gtacgattac atcagtgagc ggccacggct g 521 95 578 DNA MOUSE 95atactccagg gcagcctgtc cctgctgtat ccctgctccc aaggaaaggc atccttaaga 60agtctcgaca gcgtgaatct ggttactact cctctccaga gcccagcgag tctggggaac 120tcttagacgc cagtgatgtg ttgtgagtgg ggaccccgtg gagcagaagt ctccacaggc 180ttcagggctc ctcctccacc gcaagggcat tctcaaactc aatggcaagt tctcccgcac 240agccttagaa ggcactaccc ctagcacctt tggctccctg gaccaactgg cctcctccca 300tcctgcagcc cggccagccg cccctcaggg gctgtgagtg aggacagcat cctgtcctcc 360gagtcctttg accaattgga cttgcctgaa cgtcttcccg aaaccccact gaggggctgt 420gtgtctgtgg acaacctgag ggggcttgag cagcctccct cagaaggtct gaagcgatgg 480tggcaggaat ccttggggga tagctgcttt tctctgacag actgcaagag gtgactgcag 540ctacagacaa gccctaggaa tctgctcaaa gctcagct 578 96 548 DNA MOUSE 96cctgctgtat ccctgctccc aaggaaaggc atccttaaga agtctcgaca gcgtgaatct 60ggttactact cctctccaga gcccagcgag tctggggaac tcttagacgc cagtgatgtg 120ttgtgagtgg ggaccccgtg gagcagaagt ctccacaggc ttcagggctc ctcctccacc 180gcaagggcat tctcaaactc aatggcaagt tctcccgcac agccttagaa ggcactaccc 240ctagcacctt tggctccctg gaccaactgg ctcctcccat cctgcagccc ggcccagccg 300ccctcagggg ctgtgagtga ggacagcatc ctgtcctccg agtcctttga ccaattggac 360ttgcctgaac gtcttcccga aaccccactg aggggctgtg tgtctgtgga caacctgagg 420gggcttgagc agcctccctc agaaggtctg aagcgatggt ggcaggaatc cttgggggat 480agctgctttt ctctgacaga ctgccaagag gtgactgcag cctacagaca agccctagga 540atctgctc 548 97 588 DNA MOUSE 97 gaacaggaag ccctgcgtga gggtgggcaccctagtggtg actttggccg ggcctccatg 60 gcggactggt tacgtcgctc ctcgcgccccctcctggaga atggagccaa ggtgtgcagc 120 ttcttcaagc agcacgtgcc gggaggtggaagcactgtac ctgggctgga gcggcaacat 180 tctcttaaga agtcccgaaa ggagaacgacatggctcaaa atctgcaagg tgacccggct 240 gaggatacct cttctcgccc tggcaagagcagccttaaac ttccgaaagg cattctcaag 300 aaaaagtcct ctacctcgtc aggggaggtacaggaggacc ctcaggaact cagaccggtg 360 cctgatactc cagggcagcc tgtccctgctgtatccctgc tcccaaggaa aggcatcctt 420 aagaagtctc gacagcgtga atctggttactactcctctc cagagcccag cgagtctggg 480 gaactcttag acgccagtga tgtgtttgtgagtggggacc ccgtggagca gaagtcccca 540 caggcttcag ggctcctcct ccaccgcaagggcattctca aactcaat 588 98 331 DNA CLONE 98 acatcctggt gcatggcaccatgccctttg acgggcagga tcataaaaca ctggtgaagc 60 aaatcagtaa cggggcttaccgtgagccgc ccaagccgtc cgatgcctgt ggcctgatcc 120 ggtggctgtt aatggtgaaccccacccgtc gggccacact ggaggatgta gccagtcatt 180 ggtgggtcaa ctggggttacaccaccggag tcggggaaca ggaagccctg cgtgagggtg 240 ggcaccctag tggtgactttggccgggcct ccatggcgga ctggttacgt cgctcctcgc 300 gccccctcct ggagaatggagccaaggtgt g 331 99 164 DNA MOUSE 99 tggagacgct gggcaagggc acctacgggaaggtgaagaa ggcacgagag agctcggggc 60 gtctggtggc catcaagtcc atcaggaaagacaaaatcaa agatgagcag gatctgctgc 120 acatacggag ggagattgag atcatgtcttcactcaacca cccc 164 100 261 DNA MOUSE 100 ggagccctct ctacgcctcgcctgagatag tcaacgggaa gccctatgtg ggcccagagg 60 tggacagctg gtctctgggcgttctcctgt acatcctggt gcatggcacc atgccctttg 120 acgggcagga tcataaaacactggtgaagc aaatcagtaa cggggcttac cgtgagccgc 180 ccaagccgtc cgatgcctgtggcctgatcc ggtggctgtt aatggtgaac cccacccgtc 240 gggccacact ggaggatgta g261 101 251 PRT Homo sapiens 101 Tyr Glu Phe Leu Glu Thr Leu Gly Lys GlyThr Tyr Gly Lys Val Lys 1 5 10 15 Lys Ala Arg Glu Ser Ser Gly Arg LeuVal Ala Ile Lys Ser Ile Arg 20 25 30 Lys Asp Lys Ile Lys Asp Glu Gln AspLeu Met His Ile Arg Arg Glu 35 40 45 Ile Glu Ile Met Ser Ser Leu Asn HisPro His Ile Ile Ala Ile His 50 55 60 Glu Val Phe Glu Asn Ser Ser Lys IleVal Ile Val Met Glu Tyr Ala 65 70 75 80 Ser Arg Gly Asp Leu Tyr Asp TyrIle Ser Glu Arg Gln Gln Leu Ser 85 90 95 Glu Arg Glu Ala Arg His Phe PheArg Gln Ile Val Ser Ala Val His 100 105 110 Tyr Cys His Gln Asn Arg ValVal His Arg Asp Leu Lys Leu Glu Asn 115 120 125 Ile Leu Leu Asp Ala AsnGly Asn Ile Lys Ile Ala Asp Phe Gly Leu 130 135 140 Ser Asn Leu Tyr HisGln Gly Lys Phe Leu Gln Thr Phe Cys Gly Ser 145 150 155 160 Pro Leu TyrAla Ser Pro Glu Ile Val Asn Gly Lys Pro Tyr Thr Gly 165 170 175 Pro GluVal Asp Ser Trp Ser Leu Gly Val Leu Leu Tyr Ile Leu Val 180 185 190 HisGly Thr Met Pro Phe Asp Gly His Asp His Lys Ile Leu Val Lys 195 200 205Gln Ile Ser Asn Gly Ala Tyr Arg Glu Pro Pro Lys Pro Ser Asp Ala 210 215220 Cys Gly Leu Ile Arg Trp Leu Leu Met Val Asn Pro Thr Arg Arg Ala 225230 235 240 Thr Leu Glu Asp Val Ala Ser His Trp Trp Val 245 250 102 252PRT Homo sapiens 102 Tyr Glu Leu Gln Glu Thr Leu Gly Lys Gly Thr Tyr GlyLys Val Lys 1 5 10 15 Arg Ala Thr Glu Arg Phe Ser Gly Arg Val Val AlaIle Lys Ser Ile 20 25 30 Arg Lys Asp Lys Ile Lys Asp Glu Gln Asp Met ValHis Ile Arg Arg 35 40 45 Glu Ile Glu Ile Met Ser Ser Leu Asn His Pro HisIle Ile Ser Ile 50 55 60 Tyr Glu Val Phe Glu Asn Lys Asp Lys Ile Val IleIle Met Glu Tyr 65 70 75 80 Ala Ser Lys Gly Glu Leu Tyr Asp Tyr Ile SerGlu Arg Arg Arg Leu 85 90 95 Ser Glu Arg Glu Thr Arg His Phe Phe Arg GlnIle Val Ser Ala Val 100 105 110 His Tyr Cys His Lys Asn Gly Val Val HisArg Asp Leu Lys Leu Glu 115 120 125 Asn Ile Leu Leu Asp Asp Asn Cys AsnIle Lys Ile Ala Asp Phe Gly 130 135 140 Leu Ser Asn Leu Tyr Gln Lys AspLys Phe Leu Gln Thr Phe Cys Gly 145 150 155 160 Ser Pro Leu Tyr Ala SerPro Glu Ile Val Asn Gly Arg Pro Tyr Arg 165 170 175 Gly Pro Glu Val AspSer Trp Ala Leu Gly Val Leu Leu Tyr Thr Leu 180 185 190 Val Tyr Gly ThrMet Pro Phe Asp Gly Phe Asp His Lys Asn Leu Ile 195 200 205 Arg Gln IleSer Ser Gly Glu Tyr Arg Glu Pro Thr Gln Pro Ser Asp 210 215 220 Ala ArgGly Leu Ile Arg Trp Met Leu Met Val Asn Pro Asp Arg Arg 225 230 235 240Ala Thr Ile Glu Asp Ile Ala Asn His Trp Trp Val 245 250 103 251 PRT Homosapiens 103 Tyr Glu Phe Leu Glu Thr Leu Gly Lys Gly Thr Tyr Gly Lys ValLys 1 5 10 15 Lys Ala Arg Glu Ser Ser Gly Arg Leu Val Ala Ile Lys SerIle Arg 20 25 30 Lys Asp Lys Ile Lys Asp Glu Gln Asp Leu Leu His Ile ArgArg Glu 35 40 45 Ile Glu Ile Met Ser Ser Leu Asn His Pro His Ile Ile AlaIle His 50 55 60 Glu Val Phe Glu Asn Ser Ser Lys Ile Val Ile Val Met GluTyr Ala 65 70 75 80 Ser Arg Gly Asp Leu Tyr Asp Tyr Ile Ser Glu Arg ProArg Leu Ser 85 90 95 Glu Arg Asp Ala Arg His Phe Phe Arg Gln Ile Val SerAla Leu His 100 105 110 Tyr Cys His Gln Asn Gly Ile Val His Arg Asp LeuLys Leu Glu Asn 115 120 125 Ile Leu Leu Asp Ala Asn Gly Asn Ile Lys IleAla Asp Phe Gly Leu 130 135 140 Ser Asn Leu Tyr His Lys Gly Lys Phe LeuGln Thr Phe Cys Gly Ser 145 150 155 160 Pro Leu Tyr Ala Ser Pro Glu IleVal Asn Gly Lys Pro Tyr Val Gly 165 170 175 Pro Glu Val Asp Ser Trp SerLeu Gly Val Leu Leu Tyr Ile Leu Val 180 185 190 His Gly Thr Met Pro PheAsp Gly Gln Asp His Lys Thr Leu Val Lys 195 200 205 Gln Ile Ser Asn GlyAla Tyr Arg Glu Pro Pro Lys Pro Ser Asp Ala 210 215 220 Cys Gly Leu IleArg Trp Leu Leu Met Val Asn Pro Thr Arg Arg Ala 225 230 235 240 Thr LeuGlu Asp Val Ala Ser His Trp Trp Val 245 250 104 29 DNA ArtificialSequence primer 104 ccggatccat ggagtcggtg gccttacac 29 105 28 DNAArtificial Sequence primer 105 ccggatccct aagagttccc cagactca 28 106 21DNA Artificial Sequence primer 106 tgaggcaccg ctacgagttc c 21 107 21 DNAArtificial Sequence primer 107 accggatcag gccacaggca t 21 108 23 DNAArtificial Sequence primer 108 ccagttgacc caccaatgac tgg 23 109 15 PRTArtificial Sequence synthetic peptide substrate 109 His Met Arg Ser AlaMet Ser Gly Leu His Leu Val Lys Arg Arg 1 5 10 15

We claim:
 1. An isolated SNARK protein, selected from among the groupconsisting of: (1) the rat SNARK protein of SEQ ID NO. 1 (2) a mammalianhomolog of (1) (3) a variant of (1) or (2), and (4) a chimeric form of(1) or (2) in which a SNARK domain is exchanged with a heterologousSNARK domain, wherein said variant and said chimeric forms of SNARKretain SNARK activity and have at least 70% amino acid identity with (1)or (2).
 2. An isolated SNARK protein according to claim 1, which is therat SNARK protein of SEQ ID. NO.
 1. 3. An isolated SNARK proteinaccording to claim 1, which is the human homolog of the rat SNARKprotein of SEQ ID NO.
 1. 4. An isolated SNARK protein according to claim1, which is the murine homolog of the rat SNARK protein of SEQ ID NO. 1.5. An isolated SNARK protein according to any one of claims 1-4, indetectably labeled form.
 6. An immunogenic fragment of SNARK proteindefined in any one of claims 1-4.
 7. A detectably labeled fragment of aSNARK protein defined in any one of claims 1-4.
 8. An isolatedpolynucleotide that encodes a SNARK protein defined in any one of claims1-4.
 9. A detectably labeled polynucleotide that hybridizes with apolynucleotide according to claim 8 or with the complement thereof. 10.A vector incorporating a polynucleotide as defined in claim
 8. 11. Avector according to claim 10, wherein said vector further incorporatesexpression controlling elements linked operably with said polynucleotideto drive expression thereof in a host cell.
 12. A host cellincorporating a vector according to claim
 11. 13. A method for producinga SNARK protein, comprising the step of culturing a host cell as definedin claim
 12. 14. An antibody which binds selectively to a SNARK proteinaccording to any one of claims 1-4.
 15. A detectably labeled antibodywhich binds selectively to a SNARK protein according to any one ofclaims 1-4.
 16. A method for identifying a SNARK activity modulator,comprising the step of incubating a candidate SNARK modulator with aSNARK protein according to any one of claims 1-4 and with a SNARKsubstrate under phosphorylating conditions, and then determining whetherphosphorylation has been modulated relative to a control incubation inwhich no candidate SNARK modulator has been present.
 17. A method foridentifying a SNARK expression modulator, comprising the step ofincubation a SNARK-producing cell under conditions mediating SNARKexpression with a candidate modulator of SNARK expression, and thendetermining whether SNARK gene expression or SNARK protein productionhas occurred in the presence of said SNARK expression modulator,relative to a control incubation in which no candidate SNARK expressionmodulator has been present.
 18. A modulator of SNARK activity, wheneveridentified in accordance with the method according to claim
 16. 19. Amodulator of SNARK gene expression, whenever identified in accordancewith the method according to claim
 17. 20. A method for modulating SNARKactivity in a cell, the method comprising the step of delivering to thecell a SNARK activity modulator identified by the method according toclaim
 16. 21. A composition comprising a SNARK protein as defined in anyone of claims 1-4, and a carrier suitable for delivering the SNARKprotein to a mammal.
 22. A composition comprising a SNARK antibody, anda carrier suitable for delivering the SNARK antibody to a mammal.
 23. Amethod for inhibiting the activity of a SNARK protein as defined in anyone of claims 2-4, comprising the step of delivering to a cellexpressing said protein, an agent selected from (1) an oligonucleotideor polynucleotide that hybridizes with the endogenous polynucleotideencoding said SNARK protein to arrest the transcription or translationthereof, and (2) a polypeptide that binds to or competes with saidprotein to inhibit the phosphorylating activity thereof.
 24. A methodfor enhancing the activity of a SNARK protein as defined in any one ofclaims 2-4, comprising the step of delivering to a cell expressing saidprotein, an agent selected from (1) an expressible gene encoding saidprotein thereby to enhance the level of said protein in said cell, and(2) an amount of said protein effective to increase the presence of saidprotein in said cell thereby to enhance the protein activity.
 25. Amethod for identifying downstream targets in the SNARK signalingpathway, comprising delivering a SNARK protein, a gene coding fortherefor, or an activator of the expression of said gene to a cell ortissue or organism, and then comparing the effect of said administrationon the proteomic or genomic composition of said cell, tissue ororganism, relative to an untreated counterpart thereof, thereby toidentifying downstream targets in the SNARK signaling pathway asproteins or genes that are modulated by said administration.
 26. Asubstrate having immobilized thereon a chemical entity selected from (1)a SNARK protein as defined in claims 1-4, (2) a fragment thereof, (3) anantibody selective therefor, (4) a gene coding therefor, or (5) afragment of said gene.